The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

PubMed

Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C

2017-01-01

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

PubMed

Puy, Cristina; Tucker, Erik I; Ivanov, Ivan S; Gailani, David; Smith, Stephanie A; Morrissey, James H; Gruber, András; McCarty, Owen J T

2016-01-01

Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Cyclooxygenase-2 inhibitors modulate skin aging in a catalytic activity-independent manner

PubMed Central

Lee, Mi Eun; Kim, So Ra; Lee, Seungkoo; Jung, Yu-Jin; Choi, Sun Shim; Kim, Woo Jin

2012-01-01

It has been proposed that the pro-inflammatory catalytic activity of cyclooxygenase-2 (COX-2) plays a key role in the aging process. However, it remains unclear whether the COX-2 activity is a causal factor for aging and whether COX-2 inhibitors could prevent aging. We here examined the effect of COX-2 inhibitors on aging in the intrinsic skin aging model of hairless mice. We observed that among two selective COX-2 inhibitors and one non-selective COX inhibitor studied, only NS-398 inhibited skin aging, while celecoxib and aspirin accelerated skin aging. In addition, NS-398 reduced the expression of p53 and p16, whereas celecoxib and aspirin enhanced their expression. We also found that the aging-modulating effect of the inhibitors is closely associated with the expression of type I procollagen and caveolin-1. These results suggest that pro-inflammatory catalytic activity of COX-2 is not a causal factor for aging at least in skin and that COX-2 inhibitors might modulate skin aging by regulating the expression of type I procollagen and caveolin-1. PMID:22771771

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinical relevance of hepsin and hepatocyte growth factor activator inhibitor type 2 expression in renal cell carcinoma.

PubMed

Betsunoh, Hironori; Mukai, Shoichiro; Akiyama, Yutaka; Fukushima, Tsuyoshi; Minamiguchi, Naoki; Hasui, Yoshihiro; Osada, Yukio; Kataoka, Hiroaki

2007-04-01

Cell surface proteolysis is important for the generation of bioactive proteins mediating tumor progression. Recent studies suggest that the membrane-anchored cell surface proteinases matriptase and hepsin have significant roles in tumors. We analyzed the expression and clinical relevance of matriptase and hepsin, and their inhibitors hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) in 66 cases of conventional renal cell carcinomas (RCC). The mRNA level was evaluated in paired samples from tumor and non-tumorous renal tissues by real-time reverse transcription-polymerase chain reaction. As matriptase and hepsin potently activate the proform of hepatocyte growth factor (HGF), the expression of HGF and its receptor, c-Met, was also analyzed. Although upregulation of matriptase was observed occasionally in RCC, the expression level was not associated with prognostic parameters. Hepsin was downregulated in RCC, particularly in early stage disease, but upregulated in advanced stages. There was a trend of higher hepsin expression in RCC with distant metastasis, and Kaplan-Meier survival curves showed that high hepsin expression was associated with reduced overall survival (P<0.01, log-rank test). Moreover, multivariate analysis indicated that hepsin was an independent prognostic factor. Overexpression of HGF or c-Met also showed reduced overall survival. We also observed a tendency of low HAI-2 expression with reduced overall survival and a statistical association between high hepsin and low HAI-2 level. No associations were observed between matriptase and HAI-1 and HAI-2. Our findings suggest that the balance between hepsin and its inhibitor, HAI-2, may have prognostic value in RCC.

Insights into structure and activity of natural compound inhibitors of pneumolysin

PubMed Central

Li, Hongen; Zhao, Xiaoran; Deng, Xuming; Wang, Jianfeng; Song, Meng; Niu, Xiaodi; Peng, Liping

2017-01-01

Pneumolysin is the one of the major virulence factor of the bacterium Streptococcus pneumoniae. In previous report, it is shown that β-sitosterol, a natural compound without antimicrobial activity, is a potent antagonist of pneumolysin. Here, two new pneumolysin natural compound inhibitors, with differential activity, were discovered via haemolysis assay. To explore the key factor of the conformation for the inhibition activity, the interactions between five natural compound inhibitors with differential activity and pneumolysin were reported using molecular modelling, the potential of mean force profiles. Interestingly, it is found that incorporation of the single bond (C22-C23-C24-C25) to replace the double bond (hydrocarbon sidechain) improved the anti-haemolytic activity. In view of the molecular modelling, binding of the five inhibitors to the conserved loop region (Val372, Leu460, and Tyr461) of the cholesterol binding sites led to stable complex systems, which was consistent with the result of β-sitosterol. Owing to the single bond (C22-C23-C24-C25), campesterol and brassicasterol could form strong interactions with Val372 and show higher anti-haemolytic activity, which indicated that the single bond (C22-C23-C24-C25) in inhibitors was required for the anti-haemolytic activity. Overall, the current molecular modelling work provides a starting point for the development of rational design and higher activity pneumolysin inhibitors. PMID:28165051

Andexanet alfa to reverse the anticoagulant activity of factor Xa inhibitors: a review of design, development and potential place in therapy.

PubMed

Sartori, Michelangelo; Cosmi, Benilde

2018-04-01

Direct oral anticoagulants are associated with rates of major bleeding which are not negligible, albeit lower than those associated with vitamin K antagonists. No specific reversal agent for factor Xa (FXa) direct inhibitors is currently available for clinical use. A modified activated human FXa decoy protein, andexanet alfa, is being developed that binds FXa direct inhibitors in their active site, thus reversing their anticoagulant effect. The purpose of this article is to review the design, development and clinical trials of andexanet alfa. Andexanet alfa was shown to reverse FXa inhibitors anticoagulant activity both in thrombosis animal models, healthy volunteers and patients with acute major bleeding. Andexanet alfa has been studied in double-blind, placebo-controlled phase II and III studies. A preliminary report of the phase III study showed that an effective hemostasis was obtained after andexanet alfa infusion in the majority of the patients with acute major bleeding associated with FXa inhibitors. Additional studies are ongoing and andexanet alfa is expected to be launched in the market in the near future.

Hemodialysis in a patient with severe hemophilia A and factor VIII inhibitor.

PubMed

Gopalakrishnan, Natarajan; Usha, Thiruvengadam; Thopalan, Balasubramaniyan; Dhanapriya, Jeyachandran; Dineshkumar, Thanigachalam; Thirumalvalavan, Kaliaperumal; Sakthirajan, Ramanathan

2016-10-01

Hemophilia A is a hereditary X-linked recessive disease caused by mutations in the gene encoding factor VIII (FVIII), occurring in 1 out of 10,000 persons. Life expectancy and quality of life have dramatically improved recently in patients with hemophilia. Chronic kidney disease and need for renal replacement therapy in these patients are rare. The development of inhibitors to FVIII is the most serious complication of hemophilia and makes treatment of bleeds very challenging. We describe here a 28-year-old male patient with severe hemophilia A with presence of factor VIII inhibitor, who had end stage renal disease. Central venous access device was inserted along with infusion of factor eight inhibitor bypass activity before and after the procedure. He is currently on thrice weekly hemodialysis and doing well for 6 months without bleeding episodes. To our knowledge, hemophilia A with factor VIII inhibitor managed with hemodialysis has not been reported so far. © 2016 International Society for Hemodialysis.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

PubMed

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

2017-10-27

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Small molecule inhibitors of anthrax edema factor.

PubMed

Jiao, Guan-Sheng; Kim, Seongjin; Moayeri, Mahtab; Thai, April; Cregar-Hernandez, Lynne; McKasson, Linda; O'Malley, Sean; Leppla, Stephen H; Johnson, Alan T

2018-01-15

Anthrax is a highly lethal disease caused by the Gram-(+) bacteria Bacillus anthracis. Edema toxin (ET) is a major contributor to the pathogenesis of disease in humans exposed to B. anthracis. ET is a bipartite toxin composed of two proteins secreted by the vegetative bacteria, edema factor (EF) and protective antigen (PA). Our work towards identifying a small molecule inhibitor of anthrax edema factor is the subject of this letter. First we demonstrate that the small molecule probe 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) reacts irreversibly with EF and blocks enzymatic activity. We then show that the adenosine portion of FSBA can be replaced to provide more drug-like molecules which are up to 1000-fold more potent against EF relative to FSBA, display low cross reactivity when tested against a panel of kinases, and are nanomolar inhibitors of EF in a cell-based assay of cAMP production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Induction of Tissue Factor Pathway Inhibitor 2 by hCG Regulates Periovulatory Gene Expression and Plasmin Activity

PubMed Central

Puttabyatappa, Muraly; Al-Alem, Linah F.; Zakerkish, Farnosh; Rosewell, Katherine L.; Brännström, Mats

2017-01-01

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation. PMID:27813674

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

PubMed Central

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-ichirou; Kimura, Tadashi

2017-01-01

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer. PMID:29163796

Functional Stability of Plasminogen Activator Inhibitor-1

PubMed Central

Kuru, Pinar; Toksoy Oner, Ebru; Agirbasli, Mehmet

2014-01-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease. PMID:25386620

Induction of Tissue Factor Pathway Inhibitor 2 by hCG Regulates Periovulatory Gene Expression and Plasmin Activity.

PubMed

Puttabyatappa, Muraly; Al-Alem, Linah F; Zakerkish, Farnosh; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2017-01-01

Increased proteolytic activity is a key event that aids in breakdown of the follicular wall to permit oocyte release. How the protease activity is regulated is still unknown. We hypothesize that tissue factor pathway inhibitor 2 (TFPI2), a Kunitz-type serine protease inhibitor, plays a role in regulating periovulatory proteolytic activity as in other tissues. TFPI2 is secreted into the extracellular matrix (ECM) where it is postulated to regulate physiological ECM remodeling. The expression profile of TFPI2 during the periovulatory period was assessed utilizing a well-characterized human menstrual cycle model and a gonadotropin-primed rat model. Administration of an ovulatory dose of human chorionic gonadotropin (hCG) increased TFPI2 expression dramatically in human and rat granulosa and theca cells. This increase in Tfpi2 expression in rat granulosa cells required hCG-mediated epidermal growth factor, protein kinase A, mitogen-activated protein kinase (MAPK) 1/2, p38 MAPK and protease activated receptor 1-dependent cell signaling. A small interferingRNA-mediated knockdown of TFPI2 in rat granulosa cells resulted in increased plasmin activity in the granulosa cell conditioned media. Knockdown of TFPI2 also reduced expression of multiple genes including interleukin 6 (Il6) and amphiregulin (Areg). Overexpression of TFPI2 using an adenoviral vector partially restored the expression of Il6 and Areg in TFPI2 siRNA treated rat granulosa cells. These data support the hypothesis that TFPI2 is important for moderating plasmin activity and regulating granulosa cell gene expression during the periovulatory period. We, therefore, propose that through these actions, TFPI2 aids in the tissue remodeling taking place during follicular rupture and corpus luteum formation. Copyright © 2017 by the Endocrine Society.

Role of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in psychological stress and depression.

PubMed

Tsai, Shih-Jen

2017-12-22

Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Furthermore, the neurotrophic hypothesis of depression postulates that compromised neurotrophin brain-derived neurotrophic factor (BDNF) function is directly involved in the pathophysiology of depression. In the brain, the proteolytic cleavage of proBDNF, a BDNF precursor, to mature BDNF through plasmin represents one mechanism that can change the direction of BDNF action. We also discuss the implications of tissue-type plasminogen activator and plasminogen activator inhibitor-1 alterations as biomarkers for major depressive disorder. Using drugs that increase tissue-type plasminogen activator or decrease plasminogen activator inhibitor-1 levels may open new avenues to develop conceptually novel therapeutic strategies for depression treatment.

Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2.

PubMed

Van Huffel, S; Delaei, F; Heyninck, K; De Valck, D; Beyaert, R

2001-08-10

The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.

Plasmin-dependent proteolysis of Tissue Factor Pathway Inhibitor in a mouse model of endotoxemia

PubMed Central

Lupu, Cristina; Herlea, Oana; Tang, Haiwang; Lijnen, Roger H.; Lupu, Florea

2012-01-01

Summary Background Development of a procoagulant state in sepsis, due to aberrant expression of tissue factor (TF) and sharp decrease of its major inhibitor tissue factor pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline of TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. Objective To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (LPS) in mice. Methods Plasmin generation and TFPI activity were assayed in the lungs of mice deficient of tissue-type plasminogen activator (t-PA) or plasminogen (Plg), at 2-hrs after LPS or saline injection. Results The sharp loss of lung-associated TFPI activity at 2-hrs post LPS paralleled the abrupt increase of plasmin generation. TFPI activity was significantly retained in both t-PA-/- and Plg-/- mice, which are unable to generate plasmin. Conclusion The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications. PMID:23106863

Simultaneous expression of tissue factor and tissue factor pathway inhibitor by human monocytes. A potential mechanism for localized control of blood coagulation

PubMed Central

1994-01-01

Cells of monocytic lineage can initiate extravascular fibrin deposition via expression of blood coagulation mediators. This report is about experiments on three mechanisms with the potential to modulate monocyte- initiated coagulation. Monocyte procoagulant activity was examined as a function of lipid cofactor, protein cofactor, and specific inhibitor expression during short-term culture in vitro. Lipid cofactor activity was measured as the initial rate of factor X activation by intrinsic- pathway components, the assembly of which depends on this cofactor. Lipid cofactor activity levels changed by < 30% during 48-h culture. Protein cofactor, i.e., tissue factor (TF) antigen was measured by enzyme immunoassay. It increased from 461 pg/ml to a maximum value of 3,550 pg/ml at 24 h and remained at 70% of this value. Specific TF activity, measured as factor VII-dependent factor X activation rate, decreased from 54 to 18 nM FXa/min between 24 and 48 h. TF activity did not correlate well with either lipid cofactor or TF protein levels. In contrast, the decrease in TF activity coincided in time with maximal expression of tissue factor pathway inhibitor (TFPI) mRNA, which was determined using reverse transcriptase polymerase chain reaction (RT- PCR), and with maximal TFPI protein levels measured by immunoassay. The number of mRNA copies coding for TFPI and TF in freshly isolated blood monocytes were 46 and 20 copies/cells, respectively. These values increased to 220 and 63 copies/cell during short-term cell culture in the presence of endotoxin. Results demonstrate concomitant expression by monocytes of genes coding for both the essential protein cofactor and the specific inhibitor of the extrinsic coagulation pathway. Together with functional and antigenic analyses, they also imply that the initiation of blood clotting by extravascular monocyte/macrophages can be modulated locally by TFPI independently of plasma sources of the inhibitor. PMID:8195712

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

A Biosensor of S100A4 Metastasis Factor Activation: Inhibitor Screening and Cellular Activation Dynamics†

PubMed Central

Garrett, Sarah C.; Hodgson, Louis; Rybin, Andrew; Toutchkine, Alexei; Hahn, Klaus M.; Lawrence, David S.; Bresnick, Anne R.

2011-01-01

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor. PMID:18154362

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

Anticoagulation by factor Xa inhibitors.

PubMed

Orfeo, T; Butenas, S; Brummel-Ziedins, K E; Gissel, M; Mann, K G

2010-08-01

Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (F) Xa emerging as a promising approach. To assess anticoagulant strategies targeting FXa. A deterministic computational model of tissue factor (Tf)-initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of FXa-directed anticoagulants [an antithrombin (AT)-dependent agent, fondaparinux, and an AT-independent inhibitor, Rivaroxaban] in experimental regimens relevant to long-term (suppression of new Tf-initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. Computational representations of each anticoagulant's efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long-term and acute clinical applications, respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an FXa inhibitor. We have reported that FIXa contributes to the long-term capacity of clot-associated catalysts to restart a coagulation process, suggesting that the enhanced anti-FIXa activity of fondaparinux-AT may be critical to its success in acute settings in vivo. © 2010 International Society on Thrombosis and Haemostasis.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

PubMed Central

Nagaike, Koki; Kawaguchi, Makiko; Takeda, Naoki; Fukushima, Tsuyoshi; Sawaguchi, Akira; Kohama, Kazuyo; Setoyama, Mitsuru; Kataoka, Hiroaki

2008-01-01

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1+/+ blastocysts with Hai-1/Spint1−/− embryonic stem cells successfully generated high-chimeric Hai-1/Spint1−/− embryos (B6Hai-1−/−High) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1−/− mice was caused by placental dysfunction. However, newborn B6Hai-1−/−High mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development. PMID:18832587

Health economic review of recombinant activated factor VII for treatment of bleeding episodes in hemophilia patients with inhibitors.

PubMed

Stephens, Jennifer M; Joshi, Ashish V; Sumner, Michael; Botteman, Marc F

2007-06-01

Severe hemophilia with inhibitors is a rare disease with substantial clinical, humanistic and economic consequences. This review provides an overview of the role of recombinant activated factor VII (rFVIIa) versus plasma-derived bypassing agents for hemophilia with inhibitors and summarizes the 13 formal economic analyses (6 burden of illness and 7 comparative studies) that have been published in this indication. The findings suggest that the economic impact of rFVIIa has occurred primarily during hospitalization to manage major bleeding episodes and to allow for elective orthopedic surgeries that would not have been attempted prior to rFVIIa. Comparative analyses for on-demand treatment suggest that the total cost of treating a bleeding episode with rFVIIa may be lower than with plasma-based agents due to faster bleeding resolution, higher initial efficacy rates and avoidance of second and third lines of treatment.

Tissue factor pathway inhibitor 2 is found in skin and its C-terminal region encodes for antibacterial activity.

PubMed

Papareddy, Praveen; Kalle, Martina; Sørensen, Ole E; Lundqvist, Katarina; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

2012-01-01

Tissue factor pathway inhibitor 2 (TFPI-2) is a matrix-associated serine protease inhibitor with an enigmatic function in vivo. Here, we describe that TFPI-2 is present in fibrin of wounds and also expressed in skin, where it is up-regulated upon wounding. Neutrophil elastase cleaved TFPI-2, and a C-terminal fragment was found to bind to bacteria. Similarly, a prototypic peptide representing this C-terminal part, EDC34, bound to bacteria and bacterial lipopolysaccharide, and induced bacterial permeabilization. The peptide also induced leakage in artificial liposomes, and displayed a random coil conformation upon interactions with liposomes as well as lipopolysaccharide. EDC34 was antibacterial against both Gram-negative and Gram-positive bacteria in physiological buffer conditions. The results demonstrate that the C-terminus of TFPI-2 encodes for antimicrobial activity, and may be released during wounding.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

PubMed

Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

2013-09-01

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

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.

Small-Molecule Inhibitors of the SOX18 Transcription Factor.

PubMed

Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias

2017-03-16

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor.

PubMed

Schein, Catherine H; Chen, Deliang; Ma, Lili; Kanalas, John J; Gao, Jian; Jimenez, Maria Estrella; Sower, Laurie E; Walter, Mary A; Gilbertson, Scott R; Peterson, Johnny W

2012-11-08

Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

Lapatinib-induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors.

PubMed

Chen, Yun-Ju; Yeh, Ming-Hsin; Yu, Meng-Chieh; Wei, Ya-Ling; Chen, Wen-Shu; Chen, Jhen-Yu; Shih, Chih-Yu; Tu, Chih-Yen; Chen, Chia-Hung; Hsia, Te-Chun; Chien, Pei-Hsuan; Liu, Shu-Hui; Yu, Yung-Luen; Huang, Wei-Chien

2013-11-12

Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients.

A rhodium(III)-based inhibitor of autotaxin with antiproliferative activity.

PubMed

Kang, Tian-Shu; Wang, Wanhe; Zhong, Hai-Jing; Liang, Jia-Xin; Ko, Chung-Nga; Lu, Jin-Jian; Chen, Xiu-Ping; Ma, Dik-Lung; Leung, Chung-Hang

2017-02-01

Cancer of the skin is by far the most common of all cancers. Melanoma accounts for only about 1% of skin cancers but causes a large majority of skin cancer deaths. Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), regulates physiological and pathological functions of lysophosphatidic acid (LPA), and is thus an important therapeutic target. We synthesized ten metal-based complexes and a novel cyclometalated rhodium(III) complex 1 was identified as an ATX enzymatic inhibitor using multiple methods, including ATX enzymatic assay, thermal shift assay, western immunoblotting and so on. Protein thermal shift assays showed that 1 increased the melting temperature (T m ) of ATX by 3.5°C. 1 also reduced ATX-LPA mediated downstream survival signal pathway proteins such as ERK and AKT, and inhibited the activation of the transcription factor nuclear factor κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3). 1 also exhibited strong anti-proliferative activity against A2058 melanoma cells (IC 50 =0.58μM). Structure-activity relationship indicated that both the rhodium(III) center and the auxiliary ligands of complex 1 are important for bioactivity. 1 represents a promising scaffold for the development of small-molecule ATX inhibitors for anti-tumor applications. To our knowledge, complex 1 is the first metal-based ATX inhibitor reported to date. Rhodium complexes will have the increased attention in therapeutic and bioanalytical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

PubMed

Devy, Laetitia; Rabbani, Shafaat A; Stochl, Mark; Ruskowski, Mary; Mackie, Ian; Naa, Laurent; Toews, Mark; van Gool, Reinoud; Chen, Jie; Ley, Art; Ladner, Robert C; Dransfield, Daniel T; Henderikx, Paula

2007-11-01

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

The impact of disease activity and tumour necrosis factor-α inhibitor 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. © 2016 British Society for Immunology.

Lapatinib–induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors

PubMed Central

2013-01-01

Introduction Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Methods Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Results Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. Conclusions These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients. PMID:24216290

The role of recombinant activated factor VII in the haematological management of elective orthopaedic surgery in haemophilia A patients with inhibitors

PubMed Central

Castaman, Giancarlo

2017-01-01

The clinical profile and expectations of haemophilic patients with inhibitors have changed over the last three decades, mainly because of the prolongation of life-expectancy, often resulting in an increase of the orthopaedic burden. Recombinant activated factor VII (rFVIIa) is the most frequently used bypassing agent in haemophilia patients with inhibitors during elective orthopaedic surgery. For nearly 30 years, rFVIIa has been successfully used to control haemostasis in several major and minor surgical procedures. Clinical trials, case series, reports and surveys were progressively aimed at optimising rFVIIa usage in very demanding conditions managed in highly specialised centres. Recommendations from consensus opinions and guidelines have been provided on the basis of this clinical experience. PMID:28686157

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

PubMed

Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

2003-07-01

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Secretion imbalance between tumour necrosis factor and its inhibitor in inflammatory bowel disease

PubMed Central

Noguchi, M; Hiwatashi, N; Liu, Z; Toyota, T

1998-01-01

Background—Tumour necrosis factor (TNF) α and TNF-β are soluble ligands binding to TNF receptors with similar activities; soluble TNF receptors neutralise TNF activity by acting as inhibitors. Little is known about the cytokine/soluble receptor role in inflammatory bowel disease (IBD). 
Aims—To test the hypothesis that an imbalance in secretion between TNF and TNF inhibitors plays a role in gut inflammation in patients with IBD. 
Methods—The secretion of TNF-α, TNF-β, and soluble TNF receptors was compared in the culture supernatants of colonic biopsy specimens and isolated lamina propria mononuclear cells from patients with active colonic IBD. 
Results—Spontaneous secretion of TNF-α in involved IBD mucosa was higher than in normal control and self limited colitis mucosa. Secretion of TNF-β was higher in patients with Crohn's disease than in those with ulcerative colitis. Soluble TNF receptor in IBD mucosa inhibited TNF activity. Type 2 soluble receptor release from IBD mucosa was increased in active inflammation; release from lamina propria cells was not increased. Mucosal TNF-α production correlated with severity of disease. 
Conclusions—Results showed enhanced secretion of TNF-α but failure to release enhanced amounts of soluble TNF receptor in lamina propria mononuclear cells of patients with IBD. An imbalance in secretion between TNF and TNF inhibitor may be implicated in the pathogenesis of IBD. 

 Keywords: Crohn's disease; inflammation; mucosal immunology; soluble TNF receptor; tumour necrosis factor; ulcerative colitis PMID:10189845

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

PubMed

Ward, Richard A; Anderton, Mark J; Ashton, Susan; Bethel, Paul A; Box, Matthew; Butterworth, Sam; Colclough, Nicola; Chorley, Christopher G; Chuaqui, Claudio; Cross, Darren A E; Dakin, Les A; Debreczeni, Judit É; Eberlein, Cath; Finlay, M Raymond V; Hill, George B; Grist, Matthew; Klinowska, Teresa C M; Lane, Clare; Martin, Scott; Orme, Jonathon P; Smith, Peter; Wang, Fengjiang; Waring, Michael J

2013-09-12

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Plasmin-dependent proteolysis of tissue factor pathway inhibitor in a mouse model of endotoxemia.

PubMed

Lupu, C; Herlea, O; Tang, H; Lijnen, R H; Lupu, F

2013-01-01

The development of a procoagulant state in sepsis, owing to aberrant expression of tissue factor (TF) and a sharp decrease in the level of its major inhibitor, TF pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline in TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (lipopolysaccharide [LPS]) in mice. Plasmin generation and TFPI activity were assayed in the lungs of mice deficient in tissue-type plasminogen (Plg) activator (t-PA) or Plg, at 2 h after LPS or saline injection. The sharp loss of lung-associated TFPI activity at 2 h after LPS challenge paralleled the abrupt increase in plasmin generation. TFPI activity was significantly retained in both t-PA(-/-) and Plg(-/-) mice, which are unable to generate plasmin. The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications. © 2012 International Society on Thrombosis and Haemostasis.

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

USDA-ARS?s Scientific Manuscript database

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...

[Massive bleeding symptoms in two patients with factor V inhibitor and antiphospholipid antibodies after treatment with ciprofloxacin].

PubMed

Miesbach, Wolfgang; Voigt, Jochen; Peetz, Dirk; Scharrer, Inge

2003-06-15

The development of factor V inhibitor is very rare, especially in combination with antiphospholipid antibodies. The paper describes the course of two patients with factor V inhibitor, antiphospholipid antibodies and massive bleeding symptoms after treatment with ciprofloxacin. At that time, ciprofloxacin was the only new drug given. DIAGNOSIS AND CLINICAL COURSE: First changes of the coagulation system were detected 4 days after start of treatment. In one case, occurrence was transient, and normalization was observed after terminating ciprofloxacin treatment. The other case ended with massive muscular and visceral bleedings and cardiovascular failure. Factor V inhibitor and antiphospholipid antibodies could be demonstrated even after termination of ciprofloxacin therapy. The association of treatment with ciprofloxacin and development of factor V inhibitor and antiphospholipid antibodies is probably diagnosed to rarely. These two cases emphasize the necessity of meticulous clarification of a prolonged activated partial thromboplastin time (aPTT) and a drop in prothrombin time (PT) during and after ciprofloxacin treatment.

Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

PubMed

Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

2016-09-01

In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed. © 2016 John Wiley & Sons Ltd.

Acetylcholinesterase inhibitors rapidly activate Trk neurotrophin receptors in the mouse hippocampus

PubMed Central

Autio, Henri; Mätlik, Kert; Rantamäki, Tomi; Lindemann, Lothar; Hoener, Marius C; Chao, Moses; Arumäe, Urmas; Castrén, Eero

2014-01-01

Acetylcholinesterase inhibitors are first-line therapies for Alzheimer's disease. These drugs increase cholinergic tone in the target areas of the cholinergic neurons of the basal forebrain. Basal forebrain cholinergic neurons are dependent upon trophic support by nerve growth factor (NGF) through its neurotrophin receptor, TrkA. In the present study, we investigated whether the acetylcholinesterase inhibitors donepezil and galantamine could influence neurotrophin receptor signaling in the brain. Acute administration of donepezil (3 mg/kg, i.p.) led to the rapid autophosphorylation of TrkA and TrkB neurotrophin receptors in the adult mouse hippocampus. Similarly, galantamine dose-dependently (3, 9 mg/kg, i.p.) increased TrkA and TrkB phosphorylation in the mouse hippocampus. Both treatments also increased the phosphorylation of transcription factor CREB and tended to increase the phosphorylation of AKT kinase but did not alter the activity of MAPK42/44. Chronic treatment with galantamine (3 mg/kg, i.p., 14 days), did not induce changes in hippocampal NGF and BDNF synthesis or protein levels. Our findings show that acetylcholinesterase inhibitors are capable of rapidly activating hippocampal neurotrophin signaling and thus suggest that therapies targeting Trk signaling may already be in clinical use in the treatment of AD. PMID:21820453

Structure-activity relationship studies of chalcone leading to 3-hydroxy-4,3',4',5'-tetramethoxychalcone and its analogues as potent nuclear factor kappaB inhibitors and their anticancer activities.

PubMed

Srinivasan, Balasubramanian; Johnson, Thomas E; Lad, Rahul; Xing, Chengguo

2009-11-26

Chalcone is a privileged structure, demonstrating promising anti-inflammatory and anticancer activities. One potential mechanism is to suppress nuclear factor kappa B (NF-kappaB) activation. The structures of chalcone-based NF-kappaB inhibitors vary significantly that there is minimum information about their structure-activity relationships (SAR). This study aims to establish SAR of chalcone-based compounds to NF-kappaB inhibition, to explore the feasibility of developing simple chalcone-based potent NF-kappaB inhibitors, and to evaluate their anticancer activities. Three series of chalcones were synthesized in one to three steps with the key step being aldol condensation. These candidates demonstrated a wide range of NF-kappaB inhibitory activities, some of low micromolar potency, establishing that structural complexity is not required for NF-kappaB inhibition. Lead compounds also demonstrate potent cytotoxicity against lung cancer cells. Their cytotoxicities correlate moderately well with their NF-kappaB inhibitory activities, suggesting that suppressing NF-kappaB activation is likely responsible for at least some of the cytotoxicities. One lead compound effectively inhibits lung tumor growth with no signs of adverse side effects.

Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

PubMed

Wu, Mingyi; Wen, Dandan; Gao, Na; Xiao, Chuang; Yang, Lian; Xu, Li; Lian, Wu; Peng, Wenlie; Jiang, Jianmin; Zhao, Jinhua

2015-03-06

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Recombinant factor VIIa (eptacog alfa): a review of its use in congenital hemophilia with inhibitors, acquired hemophilia, and other congenital bleeding disorders.

PubMed

Croom, Katherine F; McCormack, Paul L

2008-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is structurally similar to human plasma-derived coagulation factor VIIa, but is manufactured using DNA biotechnology. Recombinant factor VIIa interacts with thrombin-activated platelets to produce a thrombin burst leading to accelerated fibrin clot formation localized to the site of vascular injury. It is approved in many countries for use as an intravenous hemostatic agent in patients with congenital hemophilia with inhibitors, and also for acquired hemophilia, factor VII deficiency, and Glanzmann thrombasthenia in some countries. Studies have shown it to be effective and generally well tolerated when used intravenously to treat bleeding episodes or provide hemostatic cover during surgery in patients with congenital hemophilia with inhibitors, acquired hemophilia, factor VII deficiency or Glanzmann thrombasthenia. Based on available data, its efficacy in terms of patient-assessed response may be similar to that of activated prothrombin complex concentrate (aPCC), but treatment with a single 270 microg/kg dose of recombinant factor VIIa might reduce the need for rescue therapy compared with aPCC. Recombinant factor VIIa is not immunogenic in patients with hemophilia, does not produce an anamnestic response in hemophilia patients with inhibitors, and has very low thrombogenicity. It is recommended in guidelines as the treatment of choice for bleeds in patients with hemophilia B with high-responding inhibitors and for patients with factor VII deficiency, and is also a first-line therapeutic option for high-responder hemophilia A patients with inhibitors and those with acquired hemophilia. Cost data from pharmacoeconomic analyses support its use in hemophilia patients with inhibitors. Thus, recombinant factor VIIa is a valuable treatment option for patients with these rare, but potentially serious, bleeding disorders.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

PubMed

Cui, X Y; Tinholt, M; Stavik, B; Dahm, A E A; Kanse, S; Jin, Y; Seidl, S; Sahlberg, K K; Iversen, N; Skretting, G; Sandset, P M

2016-02-01

ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may

Acquired high titre factor VIII inhibitor with underlying polyarteritis nodosa.

PubMed

Snowden, J A; Hutchings, M; Spearing, R; Patton, W N

1997-05-01

We here present the case of a 70-year-old woman referred to our unit for investigation of bleeding. Investigations confirmed a high titre acquired Factor VIII inhibitor. In association there was relapse of systemic illness associated with anti-neutrophil cytoplasmic antibodies (atypical pattern) for which she had been treated five years previously. Immunosuppression was attempted, but it failed to have an impact both on the inhibitor titre and on the underlying disorder. The patient died from multi-organ failure and massive chest hemorrhage. Post-mortem showed necrotizing vasculitis of medium sized vessels at several sites, including the kidney, consistent with a diagnosis of polyarteritis nodosa. Although it is well recognised that Factor VIII inhibitors are found in conjunction with autoimmune disorders, this case is significant in that it is the first associated with histologically proven polyarteritis nodosa type vasculitis. The case illustrates the difficulties in the investigation and management of patients with acquired high titre Factor VIII inhibitors.

[A Jehovah's Witness child with hemophilia B and factor IX inhibitors undergoing scoliosis surgery].

PubMed

Chau, Anthony; Wu, John; Ansermino, Mark; Tredwell, Stephen; Purdy, Robert

2008-01-01

To describe the successful perioperative hemostatic management of a Jehovah's Witness patient with hemophilia B and anaphylactic inhibitors to factor IX, undergoing scoliosis surgery. A 14 (1/2)-yr-old boy with severe hemophilia B who had a history of anaphylactic inhibitors to factor IX was scheduled to undergo corrective scoliosis surgery. He was initially started on epoetin alfa and iron supplementation to maximize preoperative red cell mass. Additionally, he was placed on a desensitization protocol of recombinant coagulation factor IX (rFIX) and was then treated with activated recombinant coagulation factor VII (rFVIIa) during the postoperative period. Tranexamic acid was given concomitantly. The intraoperative blood loss was approximately 350 mL. The nadir hemoglobin concentration was 111 g.L(-1) on postoperative days one and two. On postoperative day 11, the patient was stable and discharged home with a hemoglobin of 138 g.L(-1). He did not require blood transfusion and no adverse events were observed. The use of rFIX, rFVIIa, erythropoetin, iron, and tranexamic acid before, during and after scoliosis surgery may be a viable and safe option for hemophilia patients with inhibitors, who refuse blood products.

Preclinical Evaluation of MET Inhibitor INC-280 With or Without the Epidermal Growth Factor Receptor Inhibitor Erlotinib in Non–Small-Cell Lung Cancer

PubMed Central

Lara, Matthew S.; Holland, William S.; Chinn, Danielle; Burich, Rebekah A.; Lara, Primo N.; Gandara, David R.; Kelly, Karen; Mack, Philip C.

2018-01-01

The MET inhibitor INC-280 restored sensitivity to erlotinib and promoted apoptosis in non–small-cell lung cancer models rendered resistant to erlotinib by hepatocyte growth factor. Background Although the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non–small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation. Methods Four NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9. Results As a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF. Conclusion Although the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a

Discovery of a tetrahydropyrimidin-2(1H)-one derivative (TAK-442) as a potent, selective, and orally active factor Xa inhibitor.

PubMed

Fujimoto, Takuya; Imaeda, Yasuhiro; Konishi, Noriko; Hiroe, Katsuhiko; Kawamura, Masaki; Textor, Garret P; Aertgeerts, Kathleen; Kubo, Keiji

2010-05-13

Coagulation enzyme factor Xa (FXa) is a particularly promising target for the development of new anticoagulant agents. We previously reported the imidazo[1,5-c]imidazol-3-one derivative 1 as a potent and orally active FXa inhibitor. However, it was found that 1 predominantly undergoes hydrolysis upon incubation with human liver microsomes, and the human specific metabolic pathway made it difficult to predict the human pharmacokinetics. To address this issue, our synthetic efforts were focused on modification of the imidazo[1,5-c]imidazol-3-one moiety of the active metabolite 3a, derived from 1, which resulted in the discovery of the tetrahydropyrimidin-2(1H)-one derivative 5k as a highly potent and selective FXa inhibitor. Compound 5k showed no detectable amide bond cleavage in human liver microsomes, exhibited a good pharmacokinetic profile in monkeys, and had a potent antithrombotic efficacy in a rabbit model without prolongation of bleeding time. Compound 5k is currently under clinical development with the code name TAK-442.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa.

PubMed

Lu, Genmin; DeGuzman, Francis R; Hollenbach, Stanley J; Karbarz, Mark J; Abe, Keith; Lee, Gail; Luan, Peng; Hutchaleelaha, Athiwat; Inagaki, Mayuko; Conley, Pamela B; Phillips, David R; Sinha, Uma

2013-04-01

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

PubMed

Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Epidermal growth factor receptor inhibitors in cancer treatment: advances, challenges and opportunities.

PubMed

Modjtahedi, Helmout; Essapen, Sharadah

2009-11-01

Aberrant expression of the epidermal growth factor receptor (EGFR) system has been reported in a wide range of epithelial cancers. In some studies, this has also been associated with a poor prognosis and resistance to the conventional forms of therapies. These discoveries have led to the strategic development of several kinds of EGFR inhibitors, five of which have gained US Food and Drug Administration approval for the treatment of patients with non-small-cell lung cancer (gefitinib and erlotinib), metastatic colorectal cancer (cetuximab and panitumumab), head and neck (cetuximab), pancreatic cancer (erlotinib) and breast (lapatinib) cancer. Despite these advances and recent studies on the predictive value of activating EGFR mutation and KRAS mutations with response in non-small-cell lung cancer and colon cancer patients, there is currently no reliable predictive marker for response to therapy with the anti-EGFR monoclonal antibodies cetuximab and panitumumab or the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib. In particular, there has been no clear association between the expression of EGFR, determined by the US Food and Drug Administration-approved EGFR PharmDX kit, and response to the EGFR inhibitors. Here, we discuss some of the controversial data and explanatory factors as well as future studies for the establishment of more reliable markers for response to therapy with EGFR inhibitors. Such investigations should lead to the selection of a more specific subpopulation of cancer patients who benefit from therapy with EGFR inhibitors, but equally to spare those who will receive no benefit or a detrimental effect from such biological agents.

Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Zhen; Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081; Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn

2015-05-01

Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocationmore » and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.« less

Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy

PubMed Central

Yu, Tianchi

2017-01-01

Hypoxia is frequently observed in solid tumors and also one of the major obstacles for effective cancer therapies. Cancer cells take advantage of their ability to adapt hypoxia to initiate a special transcriptional program that renders them more aggressive biological behaviors. Hypoxia-inducible factors (HIFs) are the key factors that control hypoxia-inducible pathways by regulating the expression of a vast array of genes involved in cancer progression and treatment resistance. HIFs, mainly HIF-1 and -2, have become potential targets for developing novel cancer therapeutics. This article reviews the updated information in tumor HIF pathways, particularly recent advances in the development of HIF inhibitors. These inhibitors interfere with mRNA expression, protein synthesis, protein degradation and dimerization, DNA binding and transcriptional activity of HIF-1 and -2, or both. Despite efforts in the past two decades, no agents directly inhibiting HIFs have been approved for treating cancer patients. By analyzing results of the published reports, we put the perspectives at the end of the article. The therapeutic efficacy of HIF inhibitors may be improved if more efforts are devoted on developing agents that are able to simultaneously target HIF-1 and -2, increasing the penetrating capacity of HIF inhibitors, and selecting suitable patient subpopulations for clinical trials. PMID:28332352

Development of Inhibitors of the PAS-B Domain of the HIF-2α Transcription Factor

PubMed Central

Rogers, Jamie L.; Bayeh, Liela; Scheuermann, Thomas H.; Longgood, Jamie; Key, Jason; Naidoo, Jacinth; Melito, Lisa; Shokri, Cameron; Frantz, Doug E.; Bruick, Richard K.; Gardner, Kevin H.; MacMillan, John B.; Tambar, Uttam K.

2013-01-01

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity-relationship study of small molecules designed to inhibit HIF-2α–ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor/protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α–ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action. PMID:23363003

A close insight to factor VIII inhibitor in the congenital hemophilia A.

PubMed

Tabriznia-Tabrizi, Shamsoreza; Gholampour, Marzie; Mansouritorghabeh, Hassan

2016-09-01

Hemophilia A (HA) has an X-linked pattern of inheritance and is the most common of the hemorrhagic disorders. HA is caused by a decreased or deficiency of the functional clotting factor VIII (FVIII) and effects 1 in 5000-10,000 male births. The common treatment for hemophilia is replacement therapy by plasma-derived or recombinant FVIII. Approximately 20-30% of people with a severe type of HA develop an inhibitor and this phenomenon is the main challenge in the management of these patients. Genetic factors and environmental determinants contribute to inhibitor development. Here, the roles of various genetic and environmental factors such as the type of FVIII concentrate used, the number of exposure days, and peak treatment time will be discussed in detail. It seems this information is helpful for hematologists. A literature review was done in January 2016 on PubMed and Scopus using the following keywords:' h(a)emophilia A & factor VIII inhibitor', 'h(a)emophilia A & factor VIII alloantibody', 'h(a)emophilia A & inhibitor'. There was no time limitation; however, there was an English language limitation placed on the articles selected. Expert commentary: Influential genetic and environmental factors in developing inhibitors have been discussed. Most of the risk factors are related to previously untreated patients with hemophili.

Nucleolin inhibitor GroA triggers reduction in epidermal growth factor receptor activation: Pharmacological implication for glial scarring after spinal cord injury.

PubMed

Goldshmit, Yona; Schokoroy Trangle, Sari; Afergan, Fabian; Iram, Tal; Pinkas-Kramarski, Ronit

2016-09-01

Glial scarring, formed by reactive astrocytes, is one of the major impediments for regeneration after spinal cord injury (SCI). Reactive astrocytes become hypertrophic, proliferate and secrete chondroitin sulphate proteoglycans into the extracellular matrix (ECM). Many studies have demonstrated that epidermal growth factor receptors (EGFR) can mediate astrocyte reactivity after neurotrauma. Previously we showed that there is crosstalk between nucleolin and EGFR that leads to increased EGFR activation followed by increased cell proliferation. Treatment with the nucleolin inhibitor GroA (AS1411) prevented these effects in vitro and in vivo. In this study, we hypothesized that similar interactions may mediate astrogliosis after SCI. Our results demonstrate that nucleolin and EGFR interaction may play a pivotal role in mediating astrocyte proliferation and reactivity after SCI. Moreover, we demonstrate that treatment with GroA reduces EGFR activation, astrocyte proliferation and chondroitin sulphate proteoglycans secretion, therefore promoting axonal regeneration and sprouting into the lesion site. Our results identify, for the first time, a role for the interaction between nucleolin and EGFR in astrocytes after SCI, indicating that nucleolin inhibitor GroA may be used as a novel treatment after neurotrauma. A major barrier for axonal regeneration after spinal cord injury is glial scar created by reactive and proliferating astrocytes. EGFR mediate astrocyte reactivity. We showed that inhibition of nucleolin by GroA, reduces EGFR activation, which results in attenuation of astrocyte reactivity and proliferation in vivo and in vitro. EGFR, epidermal growth factor receptor. © 2016 International Society for Neurochemistry.

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors

PubMed Central

Hiroi, Toyoko; Deming, Clayton B.; Zhao, Haige; Hansen, Baranda S.; Arkenbout, Elisabeth K.; Myers, Thomas J.; McDevitt, Michael A.; Rade, Jeffrey J.

2009-01-01

Objective Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathologic thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. Methods and Results Proteasome inhibitors as a class markedly induced the expression thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-κB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. Conclusions These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects. PMID:19661484

Prognostic significance of hepatocyte growth factor activator inhibitor type 1 (HAI-1) immunoreactivity in pancreatic ductal adenocarcinoma.

PubMed

Sakugawa, Chihiro; Haruyama, Yukihiro; Tanaka, Hiroyuki; Fukushima, Tsuyoshi; Kawaguchi, Makiko; Kataoka, Hiroaki

2017-12-04

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine protease inhibitor that is expressed on the surface of epithelial cells. Evidence has suggested that decreased cell surface HAI-1 in carcinoma cells results in enhanced invasiveness. However, little is known regarding the expression of HAI-1 in pancreatic ductal adenocarcinoma (PDAC). This study aimed to analyze HAI-1 expression in PDAC and its impact on patient prognosis. HAI-1 immunohistochemistry was performed on samples from 67 PDAC cases. HAI-1 expression was increased in intraepithelial neoplasia compared to the adjacent non-neoplastic ductal epithelium. Of the 67 samples tested, 58% (39/67) of PDAC cases showed diffuse (> 75%) immunoreactivity in PDAC cells. The remaining cases showed reduced HAI-1 immunoreactivity in a substantial number of cancer cells. Although there was no correlation between HAI-1 status and tumor size, histologic grade or lymph node metastasis, diffuse HAI-1 positive cases showed longer disease-free survival (DFS; p = 0.006, log-rank test). In conclusion, HAI-1 is upregulated in pancreatic intraepithelial neoplasia and broadly expressed in PDAC cells. However, PDAC cases having areas of reduced HAI-1 immunoreactivity may show shorter DFS.

Study on the activity of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and molecular docking

NASA Astrophysics Data System (ADS)

Li, Peizhen; Tian, Yueli; Zhai, Honglin; Deng, Fangfang; Xie, Meihong; Zhang, Xiaoyun

2013-11-01

Non-purine derivatives have been shown to be promising novel drug candidates as xanthine oxidase inhibitors. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) methods including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), two 3D-QSAR models for a series of non-purine xanthine oxidase (XO) inhibitors were established, and their reliability was supported by statistical parameters. Combined 3D-QSAR modeling and the results of molecular docking between non-purine xanthine oxidase inhibitors and XO, the main factors that influenced activity of inhibitors were investigated, and the obtained results could explain known experimental facts. Furthermore, several new potential inhibitors with higher activity predicted were designed, which based on our analyses, and were supported by the simulation of molecular docking. This study provided some useful information for the development of non-purine xanthine oxidase inhibitors with novel structures.

QSAR study on the antimalarial activity of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors.

PubMed

Hou, X; Chen, X; Zhang, M; Yan, A

2016-01-01

Plasmodium falciparum, the most fatal parasite that causes malaria, is responsible for over one million deaths per year. P. falciparum dihydroorotate dehydrogenase (PfDHODH) has been validated as a promising drug development target for antimalarial therapy since it catalyzes the rate-limiting step for DNA and RNA biosynthesis. In this study, we investigated the quantitative structure-activity relationships (QSAR) of the antimalarial activity of PfDHODH inhibitors by generating four computational models using a multilinear regression (MLR) and a support vector machine (SVM) based on a dataset of 255 PfDHODH inhibitors. All the models display good prediction quality with a leave-one-out q(2) >0.66, a correlation coefficient (r) >0.85 on both training sets and test sets, and a mean square error (MSE) <0.32 on training sets and <0.37 on test sets, respectively. The study indicated that the hydrogen bonding ability, atom polarizabilities and ring complexity are predominant factors for inhibitors' antimalarial activity. The models are capable of predicting inhibitors' antimalarial activity and the molecular descriptors for building the models could be helpful in the development of new antimalarial drugs.

Recombinant factor VIIa (eptacog alfa): a pharmacoeconomic review of its use in haemophilia in patients with inhibitors to clotting factors VIII or IX.

PubMed

Lyseng-Williamson, Katherine A; Plosker, Greg L

2007-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is indicated as an intravenous haemostatic agent in haemophilia patients with inhibitors to clotting factors VIII or IX. In noncomparative trials in haemophilia patients with inhibitors, on-demand home treatment with recombinant factor VIIa was effective in controlling episodes of mild to moderate bleeding and well tolerated, with early treatment being associated with a greater rate of success and the need for fewer doses than delayed treatment. Prophylactic treatment with recombinant factor VIIa was also effective in maintaining haemostasis in patients with this indication undergoing surgery. Relative to prior treatment with plasma-derived agents, treatment with recombinant factor VIIa was associated with improvements in health-related quality of life in a cost-utility study in haemophilia patients with inhibitors in Australia. In well designed decision-model cost analyses conducted from a healthcare payer perspective in several countries, on-demand treatment with recombinant factor VIIa to control mild to moderate bleeding episodes in this patient population was predicted to be cost saving or cost neutral relative to on-demand treatment with intravenous activated prothrombin complex concentrate (aPCC). Although the acquisition cost of recombinant factor VIIa was greater than that of aPCC in some studies, the greater initial efficacy of recombinant factor VIIa than aPCC resulted in lower predicted total medical costs. Results were generally robust to plausible changes in key parameters. Orthopaedic surgery with recombinant factor VIIa to maintain haemostasis in haemophilia patients with inhibitors was generally predicted to be cost saving, relative to not having surgery, over the medium to long term in modelled cost analyses from a healthcare payer perspective in the UK and US. The initial cost of surgery was high, but the difference in costs between patients

R-268712, an orally active transforming growth factor-β type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model.

PubMed

Terashima, Hideki; Kato, Mikio; Ebisawa, Masayuki; Kobayashi, Hideki; Suzuki, Kanae; Nezu, Yoshikazu; Sada, Toshio

2014-07-05

R-268712 is a novel and specific inhibitor of activin receptor-like kinase 5 (ALK5), a transforming growth factor β (TGF-β) type I receptor. Evaluation of in vitro inhibition indicated that R-268712 is a potent and selective inhibitor of ALK5 with an IC50 of 2.5nM, an approximately 5000-fold more selectivity for ALK5 than p38 mitogen-activated protein kinase (MAPK). Oral administration of R-268712 at doses of 1, 3 and 10mg/kg also inhibited the development of renal fibrosis in a dose-dependent manner in a unilateral ureteral obstruction (UUO) model. Additionally, we evaluated the efficacy of R-268712 in a heminephrectomized anti-Thy1 glomerulonephritis model at doses of 0.3 and 1mg/kg. R-268712 reduced proteinuria and glomerulosclerosis significantly with improvement of renal function. Collectively, these results suggested that R-268712 and other ALK5 inhibitors could suppress glomerulonephritis as well as glomerulosclerosis by an inhibitory mechanism that involves suppression of TGF-β signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PubMed

Guzmán, Mauricio; Sabbione, Florencia; Gabelloni, María Laura; Vanzulli, Silvia; Trevani, Analía Silvina; Giordano, Mirta Nilda; Galletti, Jeremías Gastón

2014-09-04

To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Small molecule inhibitors block Gas6-inducible TAM activation and tumorigenicity.

PubMed

Kimani, Stanley G; Kumar, Sushil; Bansal, Nitu; Singh, Kamalendra; Kholodovych, Vladyslav; Comollo, Thomas; Peng, Youyi; Kotenko, Sergei V; Sarafianos, Stefan G; Bertino, Joseph R; Welsh, William J; Birge, Raymond B

2017-03-08

TAM receptors (Tyro-3, Axl, and Mertk) are a family of three homologous type I receptor tyrosine kinases that are implicated in several human malignancies. Overexpression of TAMs and their major ligand Growth arrest-specific factor 6 (Gas6) is associated with more aggressive staging of cancers, poorer predicted patient survival, acquired drug resistance and metastasis. Here we describe small molecule inhibitors (RU-301 and RU-302) that target the extracellular domain of Axl at the interface of the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. These inhibitors effectively block Gas6-inducible Axl receptor activation with low micromolar IC 50s in cell-based reporter assays, inhibit Gas6-inducible motility in Axl-expressing cell lines, and suppress H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model. Furthermore, using homology models and biochemical verifications, we show that RU301 and 302 also inhibit Gas6 inducible activation of Mertk and Tyro3 suggesting they can act as pan-TAM inhibitors that block the interface between the TAM Ig1 ectodomain and the Gas6 Lg domain. Together, these observations establish that small molecules that bind to the interface between TAM Ig1 domain and Gas6 Lg1 domain can inhibit TAM activation, and support the further development of small molecule Gas6-TAM interaction inhibitors as a novel class of cancer therapeutics.

Transforming growth factor β-activated kinase 1 inhibitor suppresses the proliferation in triple-negative breast cancer through TGF-β/TGFR pathway.

PubMed

Zhang, Liangyu; Fu, Zelong; Li, Xia; Tang, Haitao; Luo, Jiesi; Zhang, Dehui; Zhuang, Yongzhi; Han, Zhiyang; Yin, Mingzhu

2017-09-01

Breast cancer is one of the most invasive cancer types in female population. The functional activity of Transforming growth factor β-activated kinase 1 (TAK1) in breast cancer progression increasingly attracts attention as it provides a potential target for antibreast cancer drug development. However, the fundamental role of TAK1 for triple-negative breast cancer (TNBC) progression and the effect of potential anti-TAK1 drug candidate needs to be further evaluated. Herein, we focused on the role of TAK1 in human breast cancer cells, and we hypothesized that the inhibition of TAK1 activation can repress the growth of human TNBC cells. We found that the TAK1 is robustly activated within cancer cell population of clinic-derived TNBC samples and the human breast cancer cell lines in culture. Furthermore, we determined the effect of 5Z-7-oxozeaenol (5Z-O), a TAK1-specific small molecule inhibitor, on proliferation of human TNBC cell line. 5Z-O treatment significantly suppressed the proliferation of human TNBC cells. Collectively, these demonstrate the role of TAK1 in human breast cancer and the antiproliferate effect of TAK1 inhibitor. Our study sets the stage for further research on TAK1 as a promising target for development of anti-TNBC drugs and therapeutic strategies. © 2017 John Wiley & Sons A/S.

Development of 1-aryl-3-furanyl/thienyl-imidazopyridine templates for inhibitors against hypoxia inducible factor (HIF)-1 transcriptional activity.

PubMed

Fuse, Shinichiro; Ohuchi, Toshiaki; Asawa, Yasunobu; Sato, Shinichi; Nakamura, Hiroyuki

2016-12-15

1,3-Disubstituted-imidazopyridines were designed for developing inhibitors against HIF-1 transcriptional activity. Designed compounds were rapidly synthesized from a key aromatic scaffold via microwave-assisted Suzuki-Miyaura coupling/CH direct arylation sequence. Evaluation of ability to inhibit the hypoxia induced transcriptional activity of HIF-1 revealed that the compound 2i and 3a retained the same level of the inhibitory activity comparing with that of known inhibitor, YC-1 (1). Identified, readily accessible 1-aryl-3-furanyl/thienyl-imidazopyridine templates should be useful for future drug development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibitor development after liver transplantation in congenital factor VII deficiency.

PubMed

See, W-S Q; Chang, K-O; Cheuk, D K-L; Leung, Y-Y R; Chan, G C-F; Chan, S-C; Ha, S-Y

2016-09-01

Congenital factor VII (FVII) deficiency is the commonest type of the rare bleeding disorders. Very few cases of congenital FVII deficiency developed inhibitor and liver transplant is considered as definitive treatment. In the literature, twelve patients with congenital FVII deficiency developed inhibitors. Two had spontaneous resolution of inhibitors and one did not respond to high dose recombinant factor VIIa (rFVIIa) and died. Regarding liver transplant in congenital FVII patients, seven patients underwent liver transplant with good prognosis. We report a 5-year-old girl with confirmed severe congenital FVII deficiency since neonatal period. She suffered from recurrent intracranial bleeding despite rFVIIa replacement. After auxiliary liver transplant at the age of 4, she continued to show persistent deranged clotting profile and was found to have inhibitor towards FVII. Interestingly, she was still responsive to rFVIIa replacement. © 2016 John Wiley & Sons Ltd.

Inhibitory spectrum of alpha 2-plasmin inhibitor.

PubMed Central

Saito, H; Goldsmith, G H; Moroi, M; Aoki, N

1979-01-01

alpha 2-Plasmin inhibitor (alpha 2PI) has been recently characterized as a fast-reacting inhibitor of plasmin in human plasma and appears to play an important role in the regulation of fibrinolysis in vivo. We have studied the effect of purified alpha 2PI upon various proteases participating in human blood coagulation and kinin generation. At physiological concentration (50 microgram/ml), alpha 2PI inhibited the clot-promoting and prekallikrein-activating activity of Hageman factor fragments, the amidolytic, kininogenase, and clot-promoting activities of plasma kallikrein, and the clot-promoting properties of activated plasma thromboplastin antecedent (PTA, Factor XIa) and thrombin. alpha 2PI had minimal inhibitory effect on surface-bound activated PTA and activated Stuart factor (Factor Xa). alpha 2PI did not inhibit the activity of activated Christmas factor (Factor IXa) or urinary kallikrein. Heparin (1.5-2.0 units/ml) did not enhance the inhibitory function of alpha 2PI. These results suggest that, like other plasma protease inhibitors, alpha 2PI possesses a broad in vitro spectrum of inhibitory properties. PMID:156364

Predictors of Discontinuation of Tumor Necrosis Factor Inhibitors in Patients with Rheumatoid Arthritis

PubMed Central

AGARWAL, SANDEEP K.; GLASS, ROBERTA J.; SHADICK, NANCY A.; COBLYN, JONATHAN S.; ANDERSON, RONALD J.; MAHER, NANCY E.; WEINBLATT, MICHAEL E.; SOLOMON, DANIEL H.

2009-01-01

Objective Tumor necrosis factor-α (TNF) inhibitors have transformed management of rheumatoid arthritis (RA); however, many patients discontinue TNF inhibitors. Our goal was to determine the discontinuation rate of TNF inhibitors and identify predictors associated with discontinuation. Methods Enrollees in the Brigham RA Sequential Study (BRASS) formed the eligible cohort. Patients reporting use of a TNF inhibitor with at least 6 months of followup were followed until reporting TNF inhibitor discontinuation or their last study visit if they continued therapy. Potential predictor variables, including demographic and clinical data assessed at baseline and 6 months prior to study endpoint, were identified using a Cox proportional regression. Results Among 961 patients in BRASS, 503 were using a TNF inhibitor with at least 6 months of followup in BRASS (mean length of followup 39 mo, SD 13). Two hundred ten patients (42%) reported discontinuation of TNF inhibitor. Higher physician global scores (hazard ratio 1.27, 95% CI 1.18–1.38) and RA Disease Activity Index scores (HR 1.13, 95% CI 1.05–1.22) 6 months prior to stopping the TNF inhibitor and higher number of TNF inhibitors used previously (HR 1.30, 95% CI 1.03–1.66) were associated with discontinuation of TNF inhibitor. Prior use of synthetic disease modifying antirheumatic drugs (HR 0.50, 95% CI 0.34–0.72) and more years of cumulative methotrexate use (HR 0.24, 95% CI 0.12–0.47) were inversely associated with discontinuation of TNF inhibitor. Conclusion These data demonstrate that a significant number of patients with RA discontinue TNF inhibitors. Several easily characterized clinical variables have a modest predictive association with reduced probability of TNF inhibitor discontinuation. PMID:18634159

Changing patterns of tumor necrosis factor inhibitor use in 9074 patients with rheumatoid arthritis.

PubMed

Yazici, Yusuf; Krasnokutsky, Svetlana; Barnes, Jaime P; Hines, Patricia L; Wang, Jason; Rosenblatt, Lisa

2009-05-01

Patients with rheumatoid arthritis (RA) commonly switch between tumor necrosis factor (TNF) inhibitors after failing to control disease activity. Much of the clinical data that support switching to a second TNF agent when one agent fails to work has come from small, short-term studies. We utilized a US insurance claims database to determine patterns of use such as dose escalation, time to discontinuation, and switching between TNF inhibitors in patients with RA. A retrospective analysis was performed using an insurance claims database in the US from 2000 to 2005. TNF inhibitor use, time to switch, dose escalation, and continuation times were analyzed in patients with RA. Nine thousand seventy-four patients with RA started TNF inhibitors during the period 2000 to 2005. Etanercept was the most commonly used TNF inhibitor; infliximab had the highest duration of continuation, about 50% at 2 years. In addition, infliximab showed higher rates of dose escalation compared to etanercept and adalimumab. For all TNF inhibitors, time to switching decreased from 2000 to 2005. TNF inhibitor use patterns changed from 2000 to 2005, with more frequent changes among the different TNF inhibitors and a shorter duration of treatment before the change. Only about 50% of TNF inhibitors are still continued at 2 years, reflecting the difference between randomized clinical trials and real-world experience.

Placotylene A, an Inhibitor of the Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclast Differentiation, from a Korean Sponge Placospongia sp.

PubMed Central

Kim, Hiyoung; Kim, Kwang-Jin; Yeon, Jeong-Tae; Kim, Seong Hwan; Won, Dong Hwan; Choi, Hyukjae; Nam, Sang-Jip; Son, Young-Jin; Kang, Heonjoong

2014-01-01

A new inhibitor, placotylene A (1), of the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, and a regioisomer of placotylene A, placotylene B (2), were isolated from a Korean marine sponge Placospongia sp. The chemical structures of placotylenes A and B were elucidated on the basis of 1D and 2D NMR, along with MS spectral analysis and revealed as an iodinated polyacetylene class of natural products. Placotylene A (1) displayed inhibitory activity against RANKL-induced osteoclast differentiation at 10 μM while placotylene B (2) did not show any significant activity up to 100 μM, respectively. PMID:24705502

Recombinant factor concentrates may increase inhibitor development: a single centre cohort study.

PubMed

Strauss, T; Lubetsky, A; Ravid, B; Bashari, D; Luboshitz, J; Lalezari, S; Misgav, M; Martinowitz, U; Kenet, G

2011-07-01

Recent reports have raised concerns regarding potential risk factors for inhibitor development. In Israel, all haemophilia patients (n = 479) are followed by the National Hemophilia Center. Most children are neonatally exposed to factor concentrate (due to circumcision performed at the age of 8 days). The impact of early exposure and recombinant FVIII products (rFVIII) administration (approved in Israel since 1996) upon inhibitor occurrence in our cohort of haemophilia A (HA) patients was analysed. Two hundred ninety-two consecutive paediatric cases with a first symptomatic onset of HA were enrolled and followed over a median time of 7 years [min-max: 9 months to 17 years]. Study endpoint was inhibitor development against factor VIII. In addition, the treatment regimens applied, i.e. bolus administration or 'continuous infusion' and the family history of inhibitor development were investigated. During the follow-up period 31/292 children (10.6%) developed high titre inhibitors. Inhibitors occurred in 14/43 (32.5%) HA patients neonatally exposed to rFVIII, as compared to 22/249 previously treated with Plasma Derived (PD) products (8.8%). The odds ratio for inhibitor formation in rFVIII treated HA patients was 3.43 (95% CI: 1.36-8.65). Transient inhibitor evolved among 2/43 paediatric HA patients, only among those treated with rFVIII. The risk of inhibitor detection significantly increased among HA children treated by continuous infusion (P = 0.025). Our experience shows that the risk of inhibitor formation may be increased by early exposure to recombinant concentrates. The multiple variables affecting inhibitor incidence deserve further attention by larger prospective studies. © 2011 Blackwell Publishing Ltd.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

PubMed

Yevglevskis, Maksims; Lee, Guat L; Nathubhai, Amit; Petrova, Yoana D; James, Tony D; Threadgill, Michael D; Woodman, Timothy J; Lloyd, Matthew D

2018-04-30

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC 50  = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1*

PubMed Central

Cale, Jacqueline M.; Li, Shih-Hon; Warnock, Mark; Su, Enming J.; North, Paul R.; Sanders, Karen L.; Puscau, Maria M.; Emal, Cory D.; Lawrence, Daniel A.

2010-01-01

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1. PMID:20061381

p21-activated kinase inhibitors.

PubMed

Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Chernoff, Jonathan

2013-01-01

The p21-activated kinases (PAKs) are Ser/Thr kinases in the STE20 kinase family with important roles in regulating cytoskeletal organization, cell migration, and signaling. The PAK enzyme family comprises six members subdivided into two groups: Group I, represented by PAK1, 2, and 3, and Group II, represented by PAK 4, 5, and 6, based on sequence and structural homology. Individual PAK isoforms were found to be overexpressed and amplified in a variety of human cancers, and in vitro and in vivo studies using genetically engineered systems as well as small-molecule tool compounds have suggested therapeutic utility of PAKs as oncology targets. The identification of potent and kinome-selective ATP-competitive PAK inhibitors has proven challenging, likely caused by the openness and unique plasticity of the ATP-binding site of PAK enzymes. Progress in achieving increased kinase selectivity has been achieved with certain inhibitors but at the expense of increased molecular weight. Allosteric inhibitors, such as IPA-3, leverage the unique Group I PAK autoregulatory domain for selective inhibition, and this approach might provide an outlet to evade the kinase selectivity challenges observed with ATP-competitive PAK inhibitors. © 2013 Elsevier Inc. All rights reserved.

Prevalence of the intron 22 inversion of the factor VIII gene and inhibitor development in Polish patients with severe hemophilia A.

PubMed

Sawecka, Jadwiga; Skulimowska, Joanna; Windyga, Jerzy; Lopaciuk, Stanisław; Kościelak, Jerzy

2005-01-01

Patients with severe hemophilia A often develop inhibitors (antibodies) against transfused factor VIII. One hundred thirteen Polish patients with severe hemophilia A, who had been treated on demand with cryoprecipitate until 1992 and exclusively with factor VIII concentrates after 1995, were examined for intron 22 inversion by Southern blotting and the presence and magnitude of inhibitor activity in blood as determined by the Bethesda assay. The patients' ages ranged 4--67 years (mean: 33.7+/-12.4 years, median: 32 years). The number of patients with the inversion amounted to 57, while in 56 patients the mutation types were unknown; 47 patients had a distal and 10 patients a proximal type of inversion. Thirteen patients with inversions (22.8%) were found to have inhibitor in their blood. Most patients (14 out of 15) who developed inhibitors in the course of cryoprecipitate therapy were high responders. Conversely, 4 of 5 patients treated between 1992 and 1995 with both cryoprecipitate and intermediate-purity factor VIII concentrates were low responders. One multitransfused patient who had remained inhibitor-free on cryoprecipitate therapy developed inhibitor after receiving a large dose of factor VIII concentrate during surgery. None of these 5 patients developed inhibitors during their 12--40 years of treatment with cryoprecipitate, suggesting that it was less immunogenic than factor VIII concentrates. The prevalence of the intron 22 inversion mutation of the factor VIII gene in Polish hemophiliacs is similar to that in other European countries. Treatment regimens with either cryoprecipitate or virus-inactivated plasma-derived factor VIII concentrates may affect inhibitor formation in hemophilia A patients.

Hydrolysis of platelet-activating factor by human serum paraoxonase.

PubMed Central

Rodrigo, L; Mackness, B; Durrington, P N; Hernandez, A; Mackness, M I

2001-01-01

Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1+/-2.8 micromol/min per mg of protein with no inhibitor; 31.4+/-2.2 micromol/min per mg of protein with 100 nM inhibitor) or phenyl acetate (242.6+/-30.8 versus 240.8+/-31.5 micromol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid. PMID:11171072

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor

PubMed Central

Tang, Haiwang; Ivanciu, Lacramioara; Popescu, Narcis; Peer, Glenn; Hack, Erik; Lupu, Cristina; Taylor, Fletcher B.; Lupu, Florea

2007-01-01

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis. PMID:17640967

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.

Hepatocyte growth factor activator inhibitor type-2 (HAI-2)/SPINT2 contributes to invasive growth of oral squamous cell carcinoma cells.

PubMed

Yamamoto, Koji; Kawaguchi, Makiko; Shimomura, Takeshi; Izumi, Aya; Konari, Kazuomi; Honda, Arata; Lin, Chen-Yong; Johnson, Michael D; Yamashita, Yoshihiro; Fukushima, Tsuyoshi; Kataoka, Hiroaki

2018-02-20

Hepatocyte growth factor activator inhibitor (HAI)-1/ SPINT1 and HAI-2/ SPINT2 are membrane-anchored protease inhibitors having homologous Kunitz-type inhibitor domains. They regulate membrane-anchored serine proteases, such as matriptase and prostasin. Whereas HAI-1 suppresses the neoplastic progression of keratinocytes to invasive squamous cell carcinoma (SCC) through matriptase inhibition, the role of HAI-2 in keratinocytes is poorly understood. In vitro homozygous knockout of the SPINT2 gene suppressed the proliferation of two oral SCC (OSCC) lines (SAS and HSC3) but not the growth of a non-tumorigenic keratinocyte line (HaCaT). Reversion of HAI-2 abrogated the growth suppression. Matrigel invasion of both OSCC lines was also suppressed by the loss of HAI-2. The levels of prostasin protein were markedly increased in HAI-2-deficient cells, and knockdown of prostasin alleviated the HAI-2 loss-induced suppression of OSCC cell invasion. Therefore, HAI-2 has a pro-invasive role in OSCC cells through suppression of prostasin. In surgically resected OSCC tissues, HAI-2 immunoreactivity increased along with neoplastic progression, showing intense immunoreactivities in invasive OSCC cells. In summary, HAI-2 is required for invasive growth of OSCC cells and may contribute to OSCC progression.

Mutation of Surface Residues to Promote Crystallization of Activated Factor XI as a Complex with Benzamidine: an Essential Step for the Iterative Structure-Based Design of Factor XI Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin,L.; Pandey, P.; Babine, R.

Activated factor XI (FXIa) is a key enzyme in the amplification phase of the blood-coagulation cascade. Thus, a selective FXIa inhibitor may have lesser bleeding liabilities and provide a safe alternative for antithrombosis therapy to available drugs on the market. In a previous report, the crystal structures of the catalytic domain of FXIa (rhFXI370-607) in complex with various ecotin mutants have been described [Jin et al. (2005), Journal of Biological Chemistry 280, 4704-4712]. However, ecotin forms a matrix-like interaction with rhFXI370-607 and is impossible to displace with small-molecule inhibitors; ecotin crystals are therefore not suitable for iterative structure-based ligand design.more » In addition, rhFXI370-607 did not crystallize in the presence of small-molecule ligands. In order to obtain the crystal structure of rhFXI370-607 with a weak small-molecule ligand, namely benzamidine, several rounds of surface-residue mutation were implemented to promote crystal formation of rhFXI370-607. A quadruple mutant of rhFXI370-607 (rhFXI370-607-S434A, T475A, C482S, K437A) readily crystallized in the presence of benzamidine. The benzamidine in the preformed crystals was easily exchanged with other FXIa small-molecule inhibitors. These crystals have facilitated the structure-based design of small-molecule FXIa inhibitors.« less

Multimodal HDAC Inhibitors with Improved Anticancer Activity.

PubMed

Schobert, Rainer; Biersack, Bernhard

2018-01-01

Histone deacetylases (HDACs) play a significant role in the proliferation and dissemination of cancer and represent promising epigenetic drug targets. The HDAC inhibitor vorinostat featuring a zinc-binding hydroxamate fragment was already clinically approved. However, HDAC inhibitors containing hydroxamic acids are often hampered by acquired or intrinsic drug resistance and may lead to enhanced tumor aggressiveness. In order to overcome these drawbacks of hydroxamate HDAC inhibitors, a series of multimodal derivatives of this compound class, including such with different zinc-binding groups, was recently developed and showed promising anticancer activity. This review provides an overview of the chemistry and pleiotropic anticancer modes of action of these conceptually new HDAC inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model.

PubMed

Rajasekaran, Devaraja; Siddiq, Ayesha; Willoughby, Jennifer L S; Biagi, Jessica M; Christadore, Lisa M; Yunes, Sarah A; Gredler, Rachel; Jariwala, Nidhi; Robertson, Chadia L; Akiel, Maaged A; Shen, Xue-Ning; Subler, Mark A; Windle, Jolene J; Schaus, Scott E; Fisher, Paul B; Hansen, Ulla; Sarkar, Devanand

2015-09-22

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.

Discovery and Development of Kelch-like ECH-Associated Protein 1. Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction Inhibitors: Achievements, Challenges, and Future Directions.

PubMed

Jiang, Zheng-Yu; Lu, Meng-Chen; You, Qi-Dong

2016-12-22

The transcription factor Nrf2 is the primary regulator of the cellular defense system, and enhancing Nrf2 activity has potential usages in various diseases, especially chronic age-related and inflammatory diseases. Recently, directly targeting Keap1-Nrf2 protein-protein interaction (PPI) has been an emerging strategy to selectively and effectively activate Nrf2. This Perspective summarizes the progress in the discovery and development of Keap1-Nrf2 PPI inhibitors, including the Keap1-Nrf2 regulatory mechanisms, biochemical techniques for inhibitor identification, and approaches for identifying peptide and small-molecule inhibitors, as well as discusses privileged structures and future directions for further development of Keap1-Nrf2 PPI inhibitors.

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies.

PubMed

Sun, Junjiang; Hua, Baolai; Chen, Xiaojing; Samulski, Richard J; Li, Chengwen

2017-08-01

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10 13 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

Oral direct thrombin inhibitors or oral factor Xa inhibitors for the treatment of deep vein thrombosis.

PubMed

Robertson, Lindsay; Kesteven, Patrick; McCaslin, James E

2015-06-30

Deep vein thrombosis (DVT) is a condition in which a clot forms in the deep veins, most commonly of the leg. It occurs in approximately 1 in 1,000 people. If left untreated, the clot can travel up to the lungs and cause a potentially life-threatening pulmonary embolism (PE). Previously, a DVT was treated with the anticoagulants heparin and vitamin K antagonists. However, two forms of novel oral anticoagulants (NOACs) have been developed: oral direct thrombin inhibitors (DTI) and oral factor Xa inhibitors. The new drugs have characteristics that may be favourable over conventional treatment, including oral administration, a predictable effect, lack of frequent monitoring or re-dosing and few known drug interactions. To date, no Cochrane review has measured the effectiveness and safety of these drugs in the treatment of DVT. To assess the effectiveness of oral DTIs and oral factor Xa inhibitors for the treatment of DVT. The Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator searched the Specialised Register (last searched January 2015) and the Cochrane Register of Studies (last searched January 2015). We searched clinical trials databases for details of ongoing or unpublished studies and the reference lists of relevant articles retrieved by electronic searches for additional citations. We included randomised controlled trials in which people with a DVT confirmed by standard imaging techniques, were allocated to receive an oral DTI or an oral factor Xa inhibitor for the treatment of DVT. Two review authors (LR, JM) independently extracted the data and assessed the risk of bias in the trials. Any disagreements were resolved by discussion with the third review author (PK). We performed meta-analyses when we considered heterogeneity low. The two primary outcomes were recurrent VTE and PE. Other outcomes included all-cause mortality and major bleeding. We calculated all outcomes using an odds ratio (OR) with a 95% confidence interval (CI). We included

Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage

DTIC Science & Technology

2006-09-01

with inhibitors to factors VIII and IX, and it is ap- proved in Europe for the treatment of patients with acquired hemophilia, congenital FVII deficiency...GARY P. WRATTEN SURGICAL SYMPOSIUM Effects of Recombinant Activated Factor VII in Traumatic Nonsurgical Intracranial Hemorrhage Christopher E. White...OBJECTIVE: To determine whether treatment with recombi- nant activated factor VII (rFVIIa) will prevent progression of bleeding in nonsurgical

Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor.

PubMed

Zhou, Qing-Hui; Sumbria, Rachita; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Boado, Ruben J; Pardridge, William M

2011-11-01

Biologic tumor necrosis factor (TNF)-α inhibitors do not cross the blood-brain barrier (BBB). A BBB-penetrating TNF-α inhibitor was engineered by fusion of the extracellular domain of the type II human TNF receptor (TNFR) to the carboxyl terminus of the heavy chain of a mouse/rat chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb-TNFR fusion protein and etanercept bound human TNF-α with high affinity and K(D) values of 374 ± 77 and 280 ± 80 pM, respectively. Neuroprotection in brain in vivo after intravenous administration of the fusion protein was examined in a mouse model of Parkinson's disease. Mice were also treated with saline or a non-BBB-penetrating TNF decoy receptor, etanercept. After intracerebral injection of the nigral-striatal toxin, 6-hydroxydopamine, mice were treated every other day for 3 weeks. Treatment with the cTfRMAb-TNFR fusion protein caused an 83% decrease in apomorphine-induced rotation, a 67% decrease in amphetamine-induced rotation, a 82% increase in vibrissae-elicited forelimb placing, and a 130% increase in striatal tyrosine hydroxylase (TH) enzyme activity. In contrast, chronic treatment with etanercept, which does not cross the BBB, had no effect on neurobehavior or striatal TH enzyme activity. A bridging enzyme-linked immunosorbent assay specific for the cTfRMAb-TNFR fusion protein showed that the immune response generated in the mice was low titer. In conclusion, a biologic TNF inhibitor is neuroprotective after intravenous administration in a mouse model of neurodegeneration, providing that the TNF decoy receptor is reengineered to cross the BBB.

p21-Activated kinase inhibitors: a patent review.

PubMed

Crawford, James J; Hoeflich, Klaus P; Rudolph, Joachim

2012-03-01

The p21-activated kinase (PAK) family of serine/threonine protein kinases is activated by binding to the small (p21) GTP-binding proteins Cdc42 and Rac. The PAK family plays important roles in cytoskeletal organisation, cellular morphogenesis and survival, and members of this family have been implicated in a wide range of diseases including cancer, infectious diseases, neurological disorders and arthritis. The present review seeks to summarise recent (up to 2011) reports of small-molecule inhibitors of p21-activated kinases. Where patent applications describe activity against a broad range of kinases and no information was provided specifically on PAK inhibition, these are excluded from this review. In patents considered to be relevant, exemplary compounds were selected and highlighted based on their representation of the chemical matter claimed, potencies, structural features and subsequent disclosure of their properties. Selected information from non-patent literature was also included. A considerable amount of research has been devoted over the past 15 years to exploring the role of PAKs in a wide range of diseases, with a focus on oncology. Published PAK inhibitors are still comparatively rare and few exhibit satisfactory kinase selectivity and 'drug-like' properties. A key question is which profile, pan-PAK, group selective or isoform selective, holds the most promise from both therapeutic and safety standpoints. To investigate this question, isoform-selective, as well as kinome-selective, PAK inhibitor tool compounds will be needed. Pfizer was the first company to progress a PAK inhibitor (pan-PAK) to clinical development; it is expected that, despite the difficulties, other PAK inhibitors will soon follow.

3D-quantitative structure-activity relationship studies on benzothiadiazepine hydroxamates as inhibitors of tumor necrosis factor-alpha converting enzyme.

PubMed

Murumkar, Prashant R; Giridhar, Rajani; Yadav, Mange Ram

2008-04-01

A set of 29 benzothiadiazepine hydroxamates having selective tumor necrosis factor-alpha converting enzyme inhibitory activity were used to compare the quality and predictive power of 3D-quantitative structure-activity relationship, comparative molecular field analysis, and comparative molecular similarity indices models for the atom-based, centroid/atom-based, data-based, and docked conformer-based alignment. Removal of two outliers from the initial training set of molecules improved the predictivity of models. Among the 3D-quantitative structure-activity relationship models developed using the above four alignments, the database alignment provided the optimal predictive comparative molecular field analysis model for the training set with cross-validated r(2) (q(2)) = 0.510, non-cross-validated r(2) = 0.972, standard error of estimates (s) = 0.098, and F = 215.44 and the optimal comparative molecular similarity indices model with cross-validated r(2) (q(2)) = 0.556, non-cross-validated r(2) = 0.946, standard error of estimates (s) = 0.163, and F = 99.785. These models also showed the best test set prediction for six compounds with predictive r(2) values of 0.460 and 0.535, respectively. The contour maps obtained from 3D-quantitative structure-activity relationship studies were appraised for activity trends for the molecules analyzed. The comparative molecular similarity indices models exhibited good external predictivity as compared with that of comparative molecular field analysis models. The data generated from the present study helped us to further design and report some novel and potent tumor necrosis factor-alpha converting enzyme inhibitors.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Skin problems and EGFR-tyrosine kinase inhibitor

PubMed Central

Kozuki, Toshiyuki

2016-01-01

Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. PMID:26826719

Pyrrolo[2,3-d]pyrimidines active as Btk inhibitors.

PubMed

Musumeci, Francesca; Sanna, Monica; Greco, Chiara; Giacchello, Ilaria; Fallacara, Anna Lucia; Amato, Rosario; Schenone, Silvia

2017-12-01

Btk is a tyrosine kinase dysregulated in several B-cell malignancies and autoimmune diseases, and this has given rise to a search for Btk inhibitors. Nevertheless, only one Btk inhibitor, ibrutinib, has been approved to date, although other compounds are currently being evaluated in clinical trials or in preclinal stages. Area covered: This review, after a brief introduction on Btk and its inhibitors already in clinical trials, focusses on pyrrolo[2,3-d]pyrimidine derivatives patented in the last five years as Btk inhibitors. Indeed, the pyrrolo[2,3-d]pyrimidine scaffold, being a deaza-isostere of adenine, the nitrogenous base of ATP, is an actively pursued target for Btk inhibitors. The patent literature since 2012 have been extensively investigated, pointing out the general features of the patented compounds and, when it is possible, their mechanism of action. Expert opinion: The recently patented pyrrolo[2,3-d]pyrimidines, acting as reversible or irreversible inhibitors, showed a very interesting in vitro activity. For this reason, the development of compounds endowed with this scaffold could afford a significant impact in the search for drug candidates for the treatment of immune diseases or B-cell malignancies.

A nuclear factor kappa B-derived inhibitor tripeptide inhibits UVB-induced photoaging process.

PubMed

Oh, Jee Eun; Kim, Min Seo; Jeon, Woo-Kwang; Seo, Young Kwon; Kim, Byung-Chul; Hahn, Jang Hee; Park, Chang Seo

2014-12-01

Ultraviolet (UV) irradiation on the skin induces photoaging which is characterized by keratinocyte hyperproliferation, generation of coarse wrinkles, worse of laxity and roughness. Upon UV irradiation, nuclear factor kappa B (NF-κB) is activated which plays a key role in signaling pathway leading to inflammation cascade and this activation stimulates expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1alpha (IL-1α) and a stress response gene cyclooxygenase-2 (COX-2). In addition, activation of NF-κB up-regulates the expression of matrix metalloprotease-1 (MMP-1) and consequently collagen in dermis is degraded. In this study, the effects of a NF-κB-derived inhibitor tripeptide on the UVB-induced photoaging and inflammation were investigated in vitro and in vivo. A NF-κB-derived inhibitor tripeptide (NF-κB-DVH) was synthesized based on the sequence of dimerization region of the subunit p65 of NF-κB. Its inhibitory activity was confirmed using chromatin immunoprecipitation assay and in situ proximate ligation assay. The effects of anti-photoaging and anti-inflammation were analyzed by Enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunochemistry. NF-κB-DVH significantly decreased UV-induced expression of TNF-α, IL-1α, MMP-1 and COX-2 while increased production of type I procollagen. Results showed NF-κB-DVH had strong anti-inflammatory activity probably by inhibiting NF-κB activation pathway and suggested to be used as a novel agent for anti-photoaging. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

PubMed

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Design, synthesis, and protein crystallography of biaryltriazoles as potent tautomerase inhibitors of macrophage migration inhibitory factor.

PubMed

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

2015-03-04

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.

Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

PubMed

Wengner, Antje M; Siemeister, Gerhard; Koppitz, Marcus; Schulze, Volker; Kosemund, Dirk; Klar, Ulrich; Stoeckigt, Detlef; Neuhaus, Roland; Lienau, Philip; Bader, Benjamin; Prechtl, Stefan; Raschke, Marian; Frisk, Anna-Lena; von Ahsen, Oliver; Michels, Martin; Kreft, Bertolt; von Nussbaum, Franz; Brands, Michael; Mumberg, Dominik; Ziegelbauer, Karl

2016-04-01

Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR. ©2016 American Association for Cancer Research.

Genetic risk factors for inhibitors in haemophilia A.

PubMed

Bardi, Edit; Astermark, Jan

2015-02-01

The current most serious side effect of haemophilia treatment is inhibitor development. Significant progress has been made over the last decades to understand why this complication occurs in some patients and it seems clear that both genetic and non-genetic factors are involved. Several issues however remain to be settled. A review was undertaken to summarise some key findings regarding the current view and available data on genetic markers of potential importance within this area. The causative F8 mutation, together with the HLA class II alleles, plays a pivotal pathophysiological role in inhibitor development. The types of mutation most frequently associated with inhibitors are large deletions, nonsense mutations, inversions, small deletions/insertions without A-runs, splice-site mutations at conserved nucleotides and certain missense mutations. Regarding HLA class II allele, it has been hard to consistently identify risk alleles. Ethnicity has consistently been associated with inhibitor risk, but the causality of this has so far not been resolved. Among immune regulatory molecules, several polymorphic molecules have been suggested to be of importance. Most of these need additional studies and immune system challenges have to be fully evaluated. Inhibitor risk should be further defined, as patients in the future may be offered non-immunogenic treatments. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

NASA Astrophysics Data System (ADS)

Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; de Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

2016-11-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

PubMed

Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

2014-08-01

Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM.

Skin problems and EGFR-tyrosine kinase inhibitor.

PubMed

Kozuki, Toshiyuki

2016-04-01

Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

PubMed

Berruyer, M; Amiral, J; Ffrench, P; Belleville, J; Bastien, O; Clerc, J; Kassir, A; Estanove, S; Dechavanne, M

1993-05-01

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

Report of a factor VIII inhibitor in a patient with autoimmune lymphoproliferative syndrome.

PubMed

Fang, B S; Sneller, M C; Straus, S E; Frenkel, L; Dale, J K; Rick, M E

2000-07-01

The occurrence of factor VIII inhibitors in non-hemophilic patients is a rare event with a potentially lethal outcome. Despite its infrequent occurrence, the association of this inhibitor with multiple autoimmune diseases is well recognized. We report the case of a patient with the recently described autoimmune lymphoproliferative syndrome (ALPS) who developed an inhibitor to factor VIII. ALPS is a disease characterized by defective lymphocyte apoptosis due to inherited mutations in genes that regulate apoptosis, with the resulting enlargement of lymphoid organs and a variety of autoimmune manifestations. Published 2000 Wiley-Liss, Inc.

Oral direct thrombin inhibitors or oral factor Xa inhibitors for the treatment of pulmonary embolism.

PubMed

Robertson, Lindsay; Kesteven, Patrick; McCaslin, James E

2015-12-04

Pulmonary embolism is a potentially life-threatening condition in which a clot can travel from the deep veins, most commonly in the leg, up to the lungs. Previously, a pulmonary embolism was treated with the anticoagulants heparin and vitamin K antagonists. Recently, however, two forms of direct oral anticoagulants (DOACs) have been developed: oral direct thrombin inhibitors (DTI) and oral factor Xa inhibitors. The new drugs have characteristics that may be favourable over conventional treatment, including oral administration, a predictable effect, lack of frequent monitoring or re-dosing and few known drug interactions. To date, no Cochrane review has measured the effectiveness and safety of these drugs in the long-term treatment (minimum duration of three months) of pulmonary embolism. To assess the effectiveness of oral DTIs and oral factor Xa inhibitors for the long-term treatment of pulmonary embolism. The Cochrane Vascular Trials Search Co-ordinator searched the Specialised Register (last searched January 2015) and the Cochrane Register of Studies (last searched January 2015). Clinical trials databases were also searched for details of ongoing or unpublished studies. We searched the reference lists of relevant articles retrieved by electronic searches for additional citations. We included randomised controlled trials in which patients with a pulmonary embolism confirmed by standard imaging techniques were allocated to receive an oral DTI or an oral factor Xa inhibitor for the long-term (minimum duration three months) treatment of pulmonary embolism. Two review authors (LR, JM) independently extracted the data and assessed the risk of bias in the trials. Any disagreements were resolved by discussion with the third author (PK). We used meta-analyses when we considered heterogeneity low. The two primary outcomes were recurrent venous thromboembolism and pulmonary embolism. Other outcomes included all-cause mortality and major bleeding. We calculated all outcomes

Inhibitors in haemophilia A: a perspective on clotting factor products as a potential contributing factor.

PubMed

Mathew, P; Dinter, H; Church, N; Humphries, T J; Kulkarni, R

2016-05-01

The occurrence of a neutralizing antibody in previously untreated patients (PUPs) with haemophilia A appears to be the result of an intricate interplay of both genetic and environmental factors. Recently, the type of factor VIII (FVIII) product used in the PUPs population has been implicated as a risk factor for inhibitor development. The aim of this review was to explore in a systematic manner potential hypotheses for the product-related findings in these studies (i.e. differences in the expression system of the cell lines used to produce recombinant FVIII [rFVIII], differences in the administered antigen load or changes in clinical practice over time). Review of the available clinical studies illustrates the high degree of variability for the risk of inhibitor development for the same products across different studies. Differences in cell lines or antigen load were not found to provide a reasonable explanation. The possibility of changes in clinical practice over time and patient selection bias (i.e. the preferential use of one product over another in patients at higher risk for inhibitors) offers a potential explanation and should be carefully considered when evaluating the studies. © 2016 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

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

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

PubMed

Liao, C L; Lin, Y L; Wu, B C; Tsao, C H; Wang, M C; Liu, C I; Huang, Y L; Chen, J H; Wang, J P; Chen, L K

2001-09-01

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway.

PubMed

Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

2016-04-07

Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.

Identification of Iguratimod as an Inhibitor of Macrophage Migration Inhibitory Factor (MIF) with Steroid-sparing Potential*

PubMed Central

Bloom, Joshua; Metz, Christine; Nalawade, Saisha; Casabar, Julian; Cheng, Kai Fan; He, Mingzhu; Sherry, Barbara; Coleman, Thomas; Forsthuber, Thomas; Al-Abed, Yousef

2016-01-01

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis. PMID:27793992

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

DOE PAGES

Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; ...

2015-02-20

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. Activitymore » 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. We also show 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.« less

Plasminogen activator inhibitor-1 4G/5G polymorphism, factor V Leiden, prothrombin mutations and the risk of VTE recurrence.

PubMed

Sundquist, Kristina; Wang, Xiao; Svensson, Peter J; Sundquist, Jan; Hedelius, Anna; Larsson Lönn, Sara; Zöller, Bengt; Memon, Ashfaque A

2015-11-25

Plasminogen-activator inhibitor (PAI)-1 is an important inhibitor of the plasminogen/plasmin system. PAI-1 levels are influenced by the 4G/5G polymorphism in the PAI-1 promoter. We investigated the relationship between the PAI-1 polymorphism and VTE recurrence, and its possible modification by factor V Leiden (FVL) and prothrombin (PTM) mutations. Patients (n=1,069) from the Malmö Thrombophilia Study were followed from discontinuation of anticoagulant treatment until diagnosis of VTE recurrence or the end of the study (maximum follow-up 9.8 years). One hundred twenty-seven patients (11.9 %) had VTE recurrence. PAI-1 was genotyped by TaqMan PCR. Cox regression analysis adjusted for age, sex and acquired risk factors of VTE showed no evidence of an association between PAI-1 genotype and risk of VTE recurrence in the study population as a whole. However, by including an interaction term in the analysis we showed that FVL but not PTM modified the effect of PAI-1 genotype: patients with the 4G allele plus FVL had a higher risk of VTE recurrence [hazard ratio (HR) =2.3, 95 % confidence interval (CI) =1.5-3.3] compared to patients with the 4G allele but no FVL (reference group) or FVL irrespective of PAI-1 genotype (HR=1.8, 95 % CI=1.3-2.5). Compared to reference group, 5G allele irrespective of FVL was associated with lower risk of VTE recurrence only when compared with 4G allele together with FVL. In conclusion, FVL has a modifying effect on PAI-1 polymorphism in relation to risk of VTE recurrence. The role of PAI-1 polymorphism as a risk factor of recurrent VTE may be FVL dependent.

Model of a ternary complex between activated factor VII, tissue factor and factor IX.

PubMed

Chen, Shu-wen W; Pellequer, Jean-Luc; Schved, Jean-François; Giansily-Blaizot, Muriel

2002-07-01

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Herpes Simplex Encephalitis during Treatment with Tumor Necrosis Factor-α Inhibitors

PubMed Central

Bradford, Russell D.; Pettit, April C.; Wright, Patty W.; Mulligan, Mark J.; Moreland, Larry W.; McLain, David A.; Gnann, John W.; Bloch, Karen C.

2012-01-01

We report 3 cases of herpes simplex virus encephalitis in patients receiving tumor necrosis factor-alpha (TNF-α) inhibitors for rheumatologic disorders. Although TNF-α inhibitors have been reported to increase the risk of other infectious diseases, to our knowledge, an association between anti–TNF-α drugs and herpes simplex virus encephalitis has not been previously described. PMID:19681709

Clinical usefulness of a functional assay for the von Willebrand factor cleaving protease (ADAMTS 13) and its inhibitor in a patient with thrombotic thrombocytopenic purpura.

PubMed

Rick, M E; Austin, H; Leitman, S F; Krizek, D M; Aronson, D L

2004-02-01

Decreased von Willebrand factor cleaving protease activity (VWFCP, ADAMTS 13) leads to persistence of unusually large multimers of von Willebrand factor that bind to platelets, causing platelet aggregates, microangiopathic hemolysis, and thrombocytopenia in patients with thrombotic thrombocytopenic purpura (TTP). The clinical value of measuring ADAMTS 13 and its inhibitor is not fully defined; the case reported here illustrates the usefulness of the assay to help confirm the clinical diagnosis in a patient with other potential causes for thrombotic microangiopathy; the assay also helped in making treatment decisions. A patient with systemic lupus erythematosis (SLE) presented with fever and abdominal pain, thrombocytopenia, and anemia. Thrombotic microangiopathy was diagnosed by the appearance of schistocytes, decreasing platelet count, and evidence of hemolysis. ADAMTS 13 was decreased and an inhibitor was demonstrated in the patient's initial blood sample within 24 hr of admission. Plasma exchange was initiated, and serial assays showed increased ADAMTS 13 activity and decreased inhibitor after each plasma exchange; there was a rebound in inhibitor and a decrease in ADAMTS 13 activity prior to the next exchange that lessened over time. Increasing levels of protease activity correlated with clinical and laboratory improvement. Measurement of ADAMTS 13 activity and its inhibitor aided in the diagnosis of this complicated case of a patient with other potential causes for microangiopathic hemolysis. Subsequent levels correlated with the clinical course, and disappearance of the inhibitor indicated that long-term plasma exchange or other immunosuppressive treatment was not needed.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

PubMed Central

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

2015-01-01

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE PAGES

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

2014-12-31

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Cholinesterase inhibitors modify the activity of intrinsic cardiac neurons.

PubMed

Darvesh, Sultan; Arora, Rakesh C; Martin, Earl; Magee, David; Hopkins, David A; Armour, J Andrew

2004-08-01

Cholinesterase inhibitors used to treat the symptoms of Alzheimer's disease (AD) inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), albeit to different degrees. Because central and peripheral neurons, including intrinsic cardiac neurons located on the surface of the mammalian heart, express both BuChE and AChE, we studied spontaneously active intrinsic cardiac neurons in the pig as a model to assess the effects of inhibition of AChE compared to BuChE. Neuroanatomical experiments showed that some porcine intrinsic cardiac neurons expressed AChE and/or BuChE. Enzyme kinetic experiments with cholinesterase inhibitors, namely, donepezil, galantamine, (+/-) huperzine A, metrifonate, rivastigmine, and tetrahydroaminoacridine, demonstrated that these compounds differentially inhibited porcine AChE and BuChE. Donepezil and (+/-) huperzine A were better reversible inhibitors of AChE, and galantamine equally inhibited both the enzymes. Tetrahydroaminoacridine was a better reversible inhibitor of BuChE. Rivastigmine caused more rapid inactivation of BuChE as compared to AChE. Neurophysiological studies showed that acetylcholine and butyrylcholine increase or decrease the spontaneous activity of the intrinsic cardiac neurons. Donepezil, galantamine, (+/-) huperzine A, and tetrahydroaminoacridine changed spontaneous neuronal activity by about 30-35 impulses per minute, while rivastigmine changed it by approximately 100 impulses per minute. It is concluded that (i) inhibition of AChE and BuChE directly affects the porcine intrinsic cardiac nervous system, (ii) the intrinsic cardiac nervous system represents a suitable model for examining the effects of cholinesterase inhibitors on mammalian neurons in vivo, and (iii) the activity of intrinsic cardiac neurons may be affected by pharmacological agents that inhibit cholinesterases.

The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NFκB Transcription Factors

PubMed Central

Rauert-Wunderlich, Hilka; Siegmund, Daniela; Maier, Eduard; Giner, Tina; Bargou, Ralf C.; Wajant, Harald; Stühmer, Thorsten

2013-01-01

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. PMID:23527154

Design, synthesis and biological evaluation of pyrazolylaminoquinazoline derivatives as highly potent pan-fibroblast growth factor receptor inhibitors.

PubMed

Fan, Jun; Dai, Yang; Shao, Jingwei; Peng, Xia; Wang, Chen; Cao, Sufen; Zhao, Bin; Ai, Jing; Geng, Meiyu; Duan, Wenhu

2016-06-01

Fibroblast growth factor receptors (FGFRs) are important oncology targets due to the dysregulation of this signaling pathway in a wide variety of human cancers. We identified a series of pyrazolylaminoquinazoline derivatives as potent FGFR inhibitors with low nanomolar potency. The representative compound 29 strongly inhibited FGFR1-3 kinase activity and suppressed FGFR signaling transduction in FGFR-addicted cancer cells; FGFRs-driven cell proliferation was also strongly inhibited regardless of mechanistic complexity implicated in FGFR activation, which further confirmed that 29 was a potent pan-FGFR inhibitor. The flexibility of our structure offered the potential to preserve good affinity for mutant FGFR, which is important for developing TKIs with long-term efficacy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

PubMed

Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

2017-02-04

Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

PubMed

Wang, Shouming; Beck, Richard; Burd, Andrew; Blench, Toby; Marlin, Frederic; Ayele, Tenagne; Buxton, Stuart; Dagostin, Claudio; Malic, Maja; Joshi, Rina; Barry, John; Sajad, Mohammed; Cheung, Chiming; Shaikh, Shaheda; Chahwala, Suresh; Chander, Chaman; Baumgartner, Christine; Holthoff, Hans-Peter; Murray, Elizabeth; Blackney, Michael; Giddings, Amanda

2010-02-25

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Quantitative measurement of negligible trypsin inhibitor activity and nutrient analysis of guar meal fractions.

PubMed

Lee, Jason T; Connor-Appleton, Stacey; Haq, Akram U; Bailey, Christopher A; Cartwright, Aubrey L

2004-10-20

A complete nutrient characterization of three possible products of guar bean processing does not apparently exist in the literature. Guar meal is a high-protein byproduct produced during extraction of galactomannan gum from the guar bean. During the extraction process, two fractions are produced (germ and hull). Germ and hull fractions are usually combined to form the marketed product, guar meal. Analyses characterized the nutrient, trypsin inhibitor, and galactomannan gum content of the three guar meal byproducts to determine which fraction is more valuable as an ingredient in poultry diets. Analyses indicated that the germ fraction is most appropriate for inclusion in poultry diets. Trypsin inhibitor activity previously reported as an antigrowth factor associated with guar meal was negligible and not considered to be a significant factor limiting its use in poultry feeds. Copyright 2004 American Chemical Society

Angiotensin I-Converting Enzyme Inhibitor Activity on Egg Albumen Fermentation

PubMed Central

Nahariah, N.; Legowo, A. M.; Abustam, E.; Hintono, A.

2015-01-01

Lactobacillus plantarum is used for fermentation of fish products, meat and milk. However, the utilization of these bacteria in egg processing has not been done. This study was designed to evaluate the potential of fermented egg albumen as a functional food that is rich in angiotensin I-converting enzyme inhibitors activity (ACE-inhibitor activity) and is antihypertensive. A completely randomized design was used in this study with six durations of fermentation (6, 12, 18, 24, 30, and 36 h) as treatments. Six hundred eggs obtained from the same chicken farm were used in the experiment as sources of egg albumen. Bacteria L. plantarum FNCC 0027 used in the fermentation was isolated from cow’s milk. The parameters measured were the total bacteria, dissolved protein, pH, total acid and the activity of ACE-inhibitors. The results showed that there were significant effects of fermentation time on the parameters tested. Total bacteria increased significantly during fermentation for 6, 12, 18, and 24 h and then decreased with the increasing time of fermentation to 30 and 36 h. Soluble protein increased significantly during fermentation to 18 h and then subsequently decreased during of fermentation to 24, 30, and 36 h. The pH value decreased markedly during fermentation. The activities of ACE-inhibitor in fermented egg albumen increased during fermentation to 18 h and then decreased with the increasing of the duration of fermentation to 24, 30, and 36 h. The egg albumen which was fermented for 18 h resulted in a functional food that was rich in ACE-inhibitor activity. PMID:25715689

Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

NASA Astrophysics Data System (ADS)

Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

2015-05-01

REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

Prognostic Factors in Cholinesterase Inhibitor Poisoning.

PubMed

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

2015-09-28

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. 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. 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. 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.

Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes.

PubMed

Petrov, V; Fagard, R; Lijnen, P

2000-05-01

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

NASA Astrophysics Data System (ADS)

Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

2017-09-01

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

Effect of MCM09, an active site-directed inhibitor of factor Xa, on B16-BL6 melanoma lung colonies in mice.

PubMed

Rossi, C; Hess, S; Eckl, R W; di Lena, A; Bruno, A; Thomas, O; Poggi, A

2006-03-01

Treatment with anticoagulant drugs has shown potential inhibitory effect on tumor invasion, although the relationship with clotting inhibition was not clear. The aim of our study was to evaluate the potential antitumor activity of MCM09, a newly developed, active site-directed, small molecule inhibitor of factor Xa (FXa) [WO0216312], and to relate the findings to anticlotting potency. MCM09 (0.1-10 mg kg(-1)) or heparin (H; 10 mg kg(-1)) was injected intravenously (i.v.), with 5 x 10(4) B16-BL6 melanoma cells, in C57BL/6 mice. Mice were killed after 18 days, to count lung colonies. Ex vivo anticoagulant activity was measured by activated partial thromboplastin time (APTT) on mouse plasma. MCM09, a selective inhibitor of FXa (IC-50 = 2.4 nm against human FXa), inhibited in a dose-dependent manner B16-BL6 melanoma lung colonies in mice. Mean lung metastasis number was 20.9 +/- 4.8 in controls (n = 10), 1.2 +/- 0.4 in mice treated with H, 10 mg kg(-1) i.v. (P < 0.01), 0.9 +/- 0.3, 9.2 +/- 2.2 and 15.5 +/- 2.6 in mice treated with MCM09, at 10 (P < 0.01), 1 (P < 0.05) and 0.1 mg kg(-1) i.v. (ns), respectively. MCM09 (10 mg kg(-1) i.v.) significantly prolonged APTT (57.1 +/- 10.2 s) 30 min after i.v. injection when compared with controls (25.3 +/- 1.6 s; P < 0.05). Lung colonies were 74.2-72.6% reduced by MCM09 (10 mg kg(-1)) given 60 or 120 min before cells, but not by MCM09 given 60 min thereafter, suggesting a direct cell interaction as a mechanism underlying antitumor activity.

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

Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.

PubMed Central

O'Rourke, J.; Wang, W. P.; Donnelly, L.; Wang, E.; Kreutzer, D. L.

1987-01-01

Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK

Aromatase inhibitor associated musculoskeletal symptoms are associated with reduced physical activity among breast cancer survivors.

PubMed

Brown, Justin C; Mao, Jun J; Stricker, Carrie; Hwang, Wei-Ting; Tan, Kay-See; Schmitz, Kathryn H

2014-01-01

Physical activity (PA) has numerous health benefits for breast cancer survivors. Recent data suggest that some breast cancer survivors treated with aromatase inhibitors may experience aromatase inhibitor associated musculoskeletal symptoms. It is unknown whether aromatase inhibitor associated musculoskeletal symptoms are associated with reduced PA and what other risk factors are associated with such PA reductions. We conducted a cross-sectional study at a large university-based breast cancer clinic among breast cancer survivors prescribed an aromatase inhibitor. At routine follow-up, we surveyed participants about aromatase inhibitor associated musculoskeletal symptoms, as well as pre-aromatase inhibitor, and current, PA levels. Among 300 participants, 90 (30%) reported a reduction of PA since the initiation of aromatase inhibitor therapy. Those with aromatase inhibitor associated musculoskeletal symptoms were more likely to report decreased PA (62% versus 38%, p = 0.001) compared with those without aromatase inhibitor associated musculoskeletal symptoms. In multivariate analyses, aromatase inhibitor associated musculoskeletal symptoms (odds ratio [OR] = 2.29 [95% confidence interval [CI]: 1.36-3.86]), and body mass index (OR = 1.06 [95% CI: 1.02-1.12]) were associated with reductions in PA. In subgroup analysis among breast cancer survivors with aromatase inhibitor associated musculoskeletal symptoms, self-reported lower extremity joint pain (OR = 1.23 [95% CI: 1.00-1.50]) and impaired lower extremity physical function (OR = 1.07 [95% CI: 1.01-1.14]) were associated with reductions in PA. Breast cancer survivors with aromatase inhibitor associated musculoskeletal symptoms were more likely to report reductions in PA since initiating aromatase inhibitor therapy compared with those without aromatase inhibitor associated musculoskeletal symptoms. Our findings suggest that tailored interventions targeting lower extremity functional limitations are needed to enable

Secreted and Transmembrane Wnt Inhibitors and Activators

PubMed Central

Cruciat, Cristina-Maria; Niehrs, Christof

2013-01-01

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand–receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

The role of small molecule platelet-derived growth factor receptor (PDGFR) inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-03-01

Platelet-derived growth factor (PDGF) was discovered as a serum-derived component necessary for the growth of smooth muscle cells, fibroblasts, and glial cells. The PDGF family is a product of four gene products and consists of five dimeric isoforms: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and the PDGF-AB heterodimer. This growth factor family plays an essential role in embryonic development and in wound healing in the adult. These growth factors mediate their effects by binding to and activating their receptor protein-tyrosine kinases, which are encoded by two genes: PDGFRA and PDGFRB. The functional receptors consist of the PDGFRα/α and PDGFRβ/β homodimers and the PDGFRα/β heterodimer. Although PDGF signaling is most closely associated with mesenchymal cells, PDGFs and PDGF receptors are widely expressed in the mammalian central nervous system. The PDGF receptors contain an extracellular domain that is made up of five immunoglobulin-like domains (Ig-d1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of about 100 amino acid residues, and a carboxyterminal tail. Although uncommon, activating mutations in the genes for PDGF or PDGF receptors have been documented in various neoplasms including dermatofibrosarcoma protuberans (DFSP) and gastrointestinal stromal tumors (GIST). In most neoplastic diseases, PDGF expression and action appear to involve the tumor stroma. Moreover, this family is pro-angiogenic. More than ten PDGFRα/β multikinase antagonists have been approved by the FDA for the treatment of several neoplastic disorders and interstitial pulmonary fibrosis (www.brimr.org/PKI/PKIs.htm). Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). We used the Schr

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

PubMed

El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities.

PubMed

Doe, Chris; Bentley, Ross; Behm, David J; Lafferty, Robert; Stavenger, Robert; Jung, David; Bamford, Mark; Panchal, Terry; Grygielko, Eugene; Wright, Lois L; Smith, Gary K; Chen, Zunxuan; Webb, Christine; Khandekar, Sanjay; Yi, Tracey; Kirkpatrick, Robert; Dul, Edward; Jolivette, Larry; Marino, Joseph P; Willette, Robert; Lee, Dennis; Hu, Erding

2007-01-01

Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.

Checkpoint inhibitors in endometrial cancer: preclinical rationale and clinical activity.

PubMed

Mittica, Gloria; Ghisoni, Eleonora; Giannone, Gaia; Aglietta, Massimo; Genta, Sofia; Valabrega, Giorgio

2017-10-27

Treatment of advanced and recurrent endometrial cancer (EC) is still an unmet need for oncologists and gynecologic oncologists. The Cancer Genome Atlas Research Network (TCGA) recently provided a new genomic classification, dividing EC in four subgroups. Two types of EC, the polymerase epsilon (POLE)-ultra-mutated and the microsatellite instability-hyper-mutated (MSI-H), are characterized by a high mutation rate providing the rationale for a potential activity of checkpoint inhibitors. We analyzed all available evidence supporting the role of tumor microenvironment (TME) in EC development and the therapeutic implications offered by immune checkpoint inhibitors in this setting. We performed a review on Pubmed with Mesh keywords 'endometrial cancer' and the name of each checkpoint inhibitor discussed in the article. The same search was operated on clinicaltrial.gov to identify ongoing clinical trials exploring PD-1/PD-L1 and CTLA-4 axis in EC, particularly focusing on POLE-ultra-muted and MSI-H cancer types. POLE-ultra-mutated and MSI-H ECs showed an active TME expressing high number of neo-antigens and an elevated amount of tumor infiltrating lymphocytes (TILs). Preliminary results from a phase-1 clinical trial (KEYNOTE-028) demonstrated antitumor activity of Pembrolizumab in EC. Moreover, both Pembrolizumab and Nivolumab reported durable clinical responses in POLE-ultra-mutated patients. Immune checkpoint inhibitors are an attractive option in POLE-ultra-mutated and MSI-H ECs. Future investigations in these subgroups include combinations of checkpoints inhibitors with chemotherapy and small tyrosine kinase inhibitors (TKIs) to enhance a more robust intra-tumoral immune response.

Inhibitor development in patients receiving recombinant factor VIII (Recombinate rAHF/Bioclate): a prospective pharmacovigilance study.

PubMed

Ewenstein, B M; Gomperts, E D; Pearson, S; O'Banion, M E

2004-09-01

Clinical trials to date have not been adequately powered to assess comparatively infrequent events such as inhibitor development in previously treated patients (PTPs). Comprehensive large-scale pharmacovigilance studies can be useful for this purpose. We prospectively collected inhibitor development reports worldwide among recipients of Recombinate rAHF recombinant factor VIII (rFVIII), also formerly distributed under the product name Bioclate, for the entire postlicensure period from 1993 through 2002. To determine level of exposure to rFVIII we also compiled the Recombinate rAHF/Bioclate International Units (IU) distributed annually. To estimate inhibitor incidence separately for previously untreated or minimally treated patients (PUPs) with 1-50 exposure days and PTPs with >50 exposure days, we used haemophilia A incidence and prevalence data and pooled mean annual rFVIII consumption per PUP and PTP from international multicentre prospective clinical trials. Documented inhibitor cases totalled 89, and the total quantity of Recombinate rAHF/Bioclate rFVIII distributed was 6.48 x10(9) IU. No lot association or other clustering of inhibitor events was evident in PTPs. The incidence of all reported inhibitors, expressed as a percentage of patients treated, was 11.9% (CI: 5.05-28.0%) for PUPs when compared with 0.123% (CI: 0.030-0.512%) for PTPs. The rates for high-titre inhibitors (>5 BU) only were 5.96% (CI: 3.00-11.8%) for PUPs and 0.0554% (CI: 0.0113-0.271%) for PTPs. Thus, incidence rates for both all inhibitors and high-titre inhibitors in PTPs were 1% of the corresponding rates in PUPs. Data from prospective PUP clinical trials involving intensive active monitoring suggest that true inhibitor incidence may be approximately twice that estimated in this pharmacovigilance study. Nevertheless, inhibitor development in PTPs receiving Recombinate rAHF/Bioclate is infrequent.

Discovery of glycyrrhetinic acid as an orally active, direct inhibitor of blood coagulation factor xa.

PubMed

Jiang, Lilong; Wang, Qiong; Shen, Shu; Xiao, Tongshu; Li, Youbin

2014-03-01

Factor Xa (FXa) plays an important role in blood coagulation. This study investigated glycyrrhetinic acid, a small molecule derived from Chinese herbs, and whether it has a direct inhibitory effect on FXa to display its anticoagulant activity. Enzyme activities of FXa, plasmin, trypsin and thrombin, inhibition of FXa enzyme kinetics and plasma clotting time by glycyrrhentinic acid were performed in vitro. A rat tail-bleeding model and a rat venous stasis model were also used to evaluate in vivo tail-bleeding time and thrombus formation, respectively. Glycyrrhetinic acid in vitro directly inhibited FXa uncompetitivly with IC50 of 32.6 ± 1.24 μmol/L, and displayed 2-, 14- and 20-fold selectivity for FXa when compared to plasmin, thrombin and trypsin, respectively. The plasma clotting time was increased in a dose-dependent manner. The prothrombin time doubled (PT2), when the concentration of glycyrrhetinic acid reached 2.02 mmol/L. During in vivo experiments intragastric administration of glycyrrhetinic acid caused a dose-dependent reduction in thrombus weight on the rat venous stasis model (all P<0.05). 50 mg/kg glycyrrhetinic acid resulted in 34.8% of venous thrombus weight lost, compared to the control. In addition, 200, 300 and 400 mg/kg doses of glycyrrhetinic acid caused a moderate hemorrhagic effect in the rat tail-bleeding model by prolonging bleeding time 1.1-, 1.5- and 1.9-fold compared to the control, respectively. Glycyrrhetinic acid is a direct inhibitor of FXa that is effective by oral administration, and with further research could be used to treat blood coagulation disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of dibutyryl cyclic adenosine monophosphate on the gene expression of plasminogen activator inhibitor-1 and tissue factor in adipocytes.

PubMed

Taniguchi, Makoto; Ono, Naoko; Hayashi, Akira; Yakura, Yuwna; Takeya, Hiroyuki

2011-10-01

Hypertrophic adipocytes in obese states express the elevated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF). An increase in the intracellular concentration of cyclic adenosine monophosphate (cAMP) promotes triglyceride hydrolysis and may improve dysregulation of adipocyte metabolism. Here, we investigate the effect of dibutyryl-cAMP (a phosphodiesterase-resistant analog of cAMP) on the gene expression of PAI-1 and TF in adipocytes. Differentiated 3T3-L1 adipocytes were treated with dibutyryl-cAMP and agents that would be expected to elevate intracellular cAMP, including cilostazol (a phosphodiesterase inhibitor with anti-platelet and vasodilatory properties), isoproterenol (a beta adrenergic agonist) and forskolin (an adenylyl cyclase activator). The levels of PAI-1 and TF mRNAs were measured using real-time quantitative reverse transcription-PCR. The treatment of adipocytes with dibutyryl-cAMP resulted in the inhibition of both lipid accumulation and TF gene expression. However, PAI-1 gene expression was slightly but significantly increased by dibutyryl-cAMP. On the other hand, cilostazol inhibited the expression of PAI-1 without affecting lipid accumulation. When the adipocytes were treated with cilostazol in combination with isoproterenol or forskolin, the inhibitory effect of cilostazol on PAI-1 gene expression was counteracted, thus suggesting that inhibition by cilostazol may not be the result of intracellular cAMP accumulation by phosphodiesterase inhibition. These results suggest the implication of cAMP in regulation of the gene expression of TF and PAI-1 in adipocytes. Our findings will serve as a useful basis for further research in therapy for obesity-associated thrombosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

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

PubMed

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.

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

PubMed

Law, Ruby H P; Wu, Guojie; Leung, Eleanor W W; Hidaka, Koushi; Quek, Adam J; Caradoc-Davies, Tom T; Jeevarajah, Devadharshini; Conroy, Paul J; Kirby, Nigel M; Norton, Raymond S; Tsuda, Yuko; Whisstock, James C

2017-05-09

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO ( trans -4-aminomethylcyclohexanecarbonyl-l-tyrosine- n -octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

PubMed

Yuan, Xuan; Gavriilaki, Eleni; Thanassi, Jane A; Yang, Guangwei; Baines, Andrea C; Podos, Steven D; Huang, Yongqing; Huang, Mingjun; Brodsky, Robert A

2017-03-01

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA -null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement

A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation

PubMed Central

Lee, Nathan V.; Lira, Maruja E.; Pavlicek, Adam; Ye, Jingjing; Buckman, Dana; Bagrodia, Shubha; Srinivasa, Sreesha P.; Zhao, Yongjun; Aparicio, Samuel; Rejto, Paul A.; Christensen, James G.; Ching, Keith A.

2012-01-01

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling. PMID:22745804

Factor VIII inhibitor in a patient with mild haemophilia A and an Asn618-->Ser mutation responsive to immune tolerance induction and cyclophosphamide.

PubMed

Vlot, André J; Wittebol, Shulamiet; Strengers, Paul F W; Turenhout, Ellen A M; Voorberg, Jan; van den Berg, H Marijke; Mauser-Bunschoten, Eveline P

2002-04-01

We describe a patient with mild haemophilia A (original value of factor VIII activity 0.30 U/ml) who developed an inhibitor (36.1 Bethesda U/ml) which cross-reacted with his endogenous factor VIII. This caused a decline in basal factor VIII level (< 0.01 U/ml) and severe haemorrhagic events. Treatment to induce immune tolerance was started with factor VIII/von Willebrand factor (VWF) concentrates, but inhibitor levels increased progressively and the patient suffered serious bleeding. Cyclophosphamide was administered and, after 8 months treatment, factor VIII levels increased to 0.20 U/ml and the inhibitor could no longer be detected. Screening of his factor VIII gene revealed a missense mutation in exon 13 that predicts substitution of Asn618-->Ser in the A2 domain of factor VIII. Immunoprecipitation analysis showed that the antibodies present in the patient's plasma reacted with metabolically labelled A2 domain and, to a lesser extent, with factor VIII light chain. Inhibitory antibodies were completely neutralized by recombinant A2 domain, whereas no neutralization was observed after the addition of factor VIII light chain (A3-C1-C2) and C2 domain. More detailed analysis showed that the majority of inhibitory antibodies were directed against residues Arg484-Ile508, a previously identified binding site for factor VIII inhibitors. Our findings suggest that immune tolerance therapy and cyclophosphamide were successful in eradicating inhibitory antibodies against a common epitope on factor VIII.

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

PubMed

Das, Jagabandhu; Kimball, S David; Hall, Steven E; Han, Wen Ching; Iwanowicz, Edwin; Lin, James; Moquin, Robert V; Reid, Joyce A; Sack, John S; Malley, Mary F; Chang, Chiehying Y; Chong, Saeho; Wang-Iverson, David B; Roberts, Daniel G M; Seiler, Steven M; Schumacher, William A; Ogletree, Martin L

2002-01-07

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

PubMed Central

Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

2012-01-01

Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

Histone Deacetylase Inhibitors Repress Tumoral Expression of the Proinvasive Factor RUNX2.

PubMed

Sancisi, Valentina; Gandolfi, Greta; Ambrosetti, Davide Carlo; Ciarrocchi, Alessia

2015-05-01

Aberrant reactivation of embryonic pathways occurs commonly in cancer. The transcription factor RUNX2 plays a fundamental role during embryogenesis and is aberrantly reactivated during progression and metastasization of different types of human tumors. In this study, we attempted to dissect the molecular mechanisms governing RUNX2 expression and its aberrant reactivation. We identified a new regulatory enhancer element, located within the RUNX2 gene, which is responsible for the activation of the RUNX2 promoter and for the regulation of its expression in cancer cells. Furthermore, we have shown that treatment with the anticancer compounds histone deacetylase inhibitor (HDACi) results in a profound inhibition of RUNX2 expression, which is determined by the disruption of the transcription-activating complex on the identified enhancer. These data envisage a possible targeting strategy to counteract the oncongenic function of RUNX2 in cancer cells and provide evidence that the cytotoxic activity of HDACi in cancer is not only dependent on the reactivation of silenced oncosuppressors but also on the repression of oncogenic factors that are necessary for survival and progression. ©2015 American Association for Cancer Research.

Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2

PubMed Central

Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong

2011-01-01

Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114

Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

PubMed

Gadgeel, Shirish M; Wozniak, Antoinette

2013-07-01

Mutations in the epidermal growth factor receptor gene (EGFR) are frequently observed in non-small-cell lung cancer (NSCLC), occurring in about 40% to 60% of never-smokers and in about 17% of patients with adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have transformed therapy for patients with EGFR-mutant NSCLC and have proved superior to chemotherapy as first-line treatment for this patient group. Despite these benefits, there are currently 2 key challenges associated with EGFR inhibitor therapy for patients with NSCLC. First, only 85% to 90% of patients with the EGFR mutation derive clinical benefit from EGFR TKIs, with the remainder demonstrating innate resistance to therapy. Second, acquired resistance to EGFR TKIs inevitably occurs in patients who initially respond to therapy, with a median duration of response of about 10 months. Mutant EGFR activates various subcellular signaling cascades, including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which demonstrates maintained activity in a variety of TKI-resistant cancers. Given the fundamental role of the PI3K/Akt/mTOR pathway in tumor oncogenesis, proliferation, and survival, PI3K pathway inhibitors have emerged as a possible solution to the problem of EGFR TKI resistance. However resistance to EGFR TKIs is associated with considerable heterogeneity and complexity. Preclinical experiments investigating these phenomena suggest that in some patients, PI3K inhibitors will have to be paired with other targeted agents if they are to be effective. This review discusses the preclinical data supporting PI3K/Akt/mTOR pathway inhibitor combinations in EGFR TKI-resistant NSCLC from the perspective of the various agents currently being investigated in clinical trials. Copyright © 2013 Elsevier Inc. All rights reserved.

X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors

PubMed Central

Wu, Guojie; Leung, Eleanor W. W.; Hidaka, Koushi; Quek, Adam J.; Caradoc-Davies, Tom T.; Jeevarajah, Devadharshini; Kirby, Nigel M.; Norton, Raymond S.; Tsuda, Yuko; Whisstock, James C.

2017-01-01

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. PMID:29296720

Synthesis and biological evaluation of kresoxim-methyl analogues as novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in cancer cells.

PubMed

Lee, Sanghyuck; Kwon, Oh Seok; Lee, Chang-Soo; Won, Misun; Ban, Hyun Seung; Ra, Choon Sup

2017-07-01

We designed and synthesized strobilurin analogues as hypoxia-inducible factor (HIF) inhibitors based on the molecular structure of kresoxim-methyl. Biological evaluation in human colorectal cancer HCT116 cells showed that most of the synthesized kresoxim-methyl analogues possessed moderate to potent inhibitory activity against hypoxia-induced HIF-1 transcriptional activation. Three candidates, compounds 11b, 11c, and 11d were identified as potent inhibitors against HIF-1 activation with IC 50 values of 0.60-0.94µM. Under hypoxic condition, compounds 11b, 11c, and 11d increased the intracellular oxygen contents, thereby attenuating the hypoxia-induced accumulation of HIF-1α protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fibroblast Growth Factor 2: An Epithelial Ductal Cell Growth Inhibitor That Drops Out in Breast Cancer

DTIC Science & Technology

2009-10-01

AD_________________ Award Number: W81XWH-08-1-0708 TITLE: Fibroblast Growth Factor 2: an...September 2008 – 14 September 2009 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fibroblast Growth Factor 2: an Epithelial Ductal Cell Growth Inhibitor...9 Fibroblast Growth Factor -2: an Epithelial Ductal Cell Growth Inhibitor that Drops Out in Breast Cancer

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

PubMed

Yoshioka, Takahiro; Shien, Kazuhiko; Namba, Kei; Torigoe, Hidejiro; Sato, Hiroki; Tomida, Shuta; Yamamoto, Hiromasa; Asano, Hiroaki; Soh, Junichi; Tsukuda, Kazunori; Nagasaka, Takeshi; Fujiwara, Toshiyoshi; Toyooka, Shinichi

2018-04-01

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2-positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2-amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin-like growth factor-1 receptor (IGF-1R), were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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

NASA Astrophysics Data System (ADS)

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

2016-07-01

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.

Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Inhibitors and Their Potential for Targeting Central Nervous System Diseases.

PubMed

Pallesen, Jakob S; Tran, Kim T; Bach, Anders

2018-05-29

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has a protective effect against oxidative stress and plays a major role in inflammation and central nervous system (CNS) diseases. Inhibition of the protein-protein interaction (PPI) between Nrf2 and its repressor protein, Kelch-like ECH-associated protein 1 (Keap1), leads to translocation of Nrf2 from the cytosol to the nucleus and expression of detoxifying antioxidant enzymes. To date, several non-covalent small-molecule Keap1-Nrf2 inhibitors have been identified; however, many of them contain carboxylic acids and are rather large in size, which likely prevents or decreases CNS permeability. This Perspective describes current small-molecule Keap1-Nrf2 inhibitors with experimental evidence for the ability to inhibit the Keap1-Nrf2 interaction by binding to Keap1 in a non-covalent manner. Binding data, biostructural studies, and biological activity are summarized for the inhibitors, and their potential as CNS tool compounds is discussed by analyzing physicochemical properties, including CNS multiparameter optimization (MPO) scoring algorithms. Finally, several strategies for identifying CNS-targeting Keap1 inhibitors are described.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laufersweiler, Matthew; Brugel, Todd; Clark, Michael

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.

MET inhibitor PHA-665752 suppresses the hepatocyte growth factor-induced cell proliferation and radioresistance in nasopharyngeal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Tongxin; Li, Qi; Sun, Quanquan

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 mediatedmore » 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.« less

THE BTK INHIBITOR PCI-32765 SYNERGISTICALLY INCREASES PROTEASOME INHIBITOR ACTIVITY IN DLBCL AND MCL CELLS SENSITIVE OR RESISTANT TO BORTEZOMIB

PubMed Central

Dasmahapatra, Girija; Patel, Hiral; Dent, Paul; Fisher, Richard I.; Friedberg, Jonathan; Grant, Steven

2012-01-01

Summary Interactions between the Bruton tyrosine kinase (BTK) inhibitor PCI-32765 and the proteasome inhibitor (bortezomib) were examined in diffuse large-B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells, including those highly resistant to bortezomib. Co-administration of PCI-32765/bortezomib synergistically increased mitochondrial injury and apoptosis in germinal centre- or activated B-cell-like-DLBCL cells and in MCL cells. These events were accompanied by marked AKT and nuclear factor (NF)-κB (NFKB1) inactivation, down-regulation of Mcl-1 (MCL1), Bcl-xL (BCL2L1), and XIAP, and enhanced DNA damage (e.g., γH2A.X formation) and endoplasmic reticulum (ER) stress. Similar interactions were observed in highly bortezomib-resistant DLBCL and MCL cells, and in primary DLBCL cells. In contrast, PCI-32765/bortezomib regimens displayed minimal toxicity toward normal CD34+ bone marrow cells. Transfection of DLBCL cells with a constitutively active AKT construct attenuated AKT inactivation and significantly diminished cell death, whereas expression of an NF-κB “super-repressor” (IκBαser34/36) increased both PCI-32765 and bortezomib lethality. Moreover, cells in which the ER stress response was disabled by a dominant-negative eIF2α construct were resistant to this regimen. Finally, combined exposure to PCI-32765 and bortezomib resulted in more pronounced and sustained reactive oxygen species (ROS) generation, and ROS scavengers significantly diminished lethality. Given promising early clinical results for PCI-32765 in DLBCL and MCL, a strategy combining BTK/ proteasome inhibitor warrants attention in these malignancies. PMID:23360303

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

PubMed Central

Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

2016-01-01

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

PubMed

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-07-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

Ten-year experience of recombinant activated factor VII use in surgical patients with congenital haemophilia with inhibitors or acquired haemophilia in Japan.

PubMed

Takedani, H; Shima, M; Horikoshi, Y; Koyama, T; Fukutake, K; Kuwahara, M; Ishiguro, N

2015-05-01

Patients with congenital haemophilia with inhibitors or acquired haemophilia are at risk of bleeding complications during surgery. In these patients, replacement therapy for the missing coagulation factor is ineffective, and a bypassing agent such as recombinant activated factor VII (rFVIIa) is required to manage bleeding. To evaluate the safety and haemostatic efficacy of rFVIIa treatment in Japanese patients with congenital haemophilia with inhibitors to FVIII/FIX or acquired haemophilia undergoing surgery. Postmarketing surveillance data from May 2000 to March 2010 were analysed to assess the haemostatic efficacy of 38 procedures in 22 patients with congenital haemophilia A, 13 procedures in seven patients with congenital haemophilia B, and five procedures in five patients with acquired haemophilia. Postoperative bleeding control was judged to be effective (bleeding was stopped completely or reduced considerably) for 34/38 procedures (89%) in patients with congenital haemophilia A, 10/13 procedures (77%) in patients with congenital haemophilia B, and 4/5 procedures (80%) in patients with acquired haemophilia. Tranexamic acid was used concomitantly for 36/56 procedures (64%). Safety was analysed for 66 procedures in 37 patients. Adverse effects potentially related to rFVIIa treatment included mild superficial thrombophlebitis, mild decrease in platelet count, and mild elevation of the serum alanine transaminase level in one patient each. All adverse effects resolved without treatment. Administration of rFVIIa provided adequate haemostasis without serious adverse effects in the majority of cases. The efficacy and safety data in Japanese patients were similar to previously published data from other countries. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

PubMed

An, S F; Fleming, K A

1991-11-01

A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.

Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

PubMed Central

Yun, Thomas H.; Cott, Jessica E.; Tapping, Richard I.; Slauch, James M.

2009-01-01

The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. PMID:18988866

Activation of Hif1α by the Prolylhydroxylase Inhibitor Dimethyoxalyglycine Decreases Radiosensitivity

PubMed Central

Ayrapetov, Marina K.; Zhu, Kaya; Parmar, Kalindi; D'Andrea, Alan D.; Price, Brendan D.

2011-01-01

Hypoxia inducible factor 1α (Hif1α) is a stress responsive transcription factor, which regulates the expression of genes required for adaption to hypoxia. Hif1α is normally hydroxylated by an oxygen-dependent prolylhydroxylase, leading to degradation and clearance of Hif1α from the cell. Under hypoxic conditions, the activity of the prolylhydroxylase is reduced and Hif1α accumulates. Hif1α is also constitutively expressed in tumor cells, where it is associated with resistance to ionizing radiation. Activation of the Hif1α transcriptional regulatory pathway may therefore function to protect normal cells from DNA damage caused by ionizing radiation. Here, we utilized the prolylhydroxylase inhibitor dimethyloxalylglycine (DMOG) to elevate Hif1α levels in mouse embryonic fibroblasts (MEFs) to determine if DMOG could function as a radioprotector. The results demonstrate that DMOG increased Hif1α protein levels and decreased the sensitivity of MEFs to ionizing radiation. Further, the ability of DMOG to function as a radioprotector required Hif1α, indicating a key role for Hif1α's transcriptional activity. DMOG also induced the Hif1α -dependent accumulation of several DNA damage response proteins, including CHD4 and MTA3 (sub-units of the NuRD deacetylase complex) and the Suv39h1 histone H3 methyltransferase. Depletion of Suv39h1, but not CHD4 or MTA3, reduced the ability of DMOG to protect cells from radiation damage, implicating increased histone H3 methylation in the radioprotection of cells. Finally, treatment of mice with DMOG prior to total body irradiation resulted in significant radioprotection of the mice, demonstrating the utility of DMOG and related prolylhydroxylase inhibitors to protect whole organisms from ionizing radiation. Activation of Hif1α through prolylhydroxylase inhibition therefore identifies a new pathway for the development of novel radiation protectors. PMID:22016813

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

PubMed

Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

2016-10-01

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

STAT inhibitors for cancer therapy

PubMed Central

2013-01-01

Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725

Heterodimeric JAK-STAT Activation as a Mechanism of Persistence to JAK2 Inhibitor Therapy

PubMed Central

Koppikar, Priya; Bhagwat, Neha; Kilpivaara, Outi; Manshouri, Taghi; Adli, Mazhar; Hricik, Todd; Liu, Fan; Saunders, Lindsay M.; Mullally, Ann; Abdel-Wahab, Omar; Leung, Laura; Weinstein, Abby; Marubayashi, Sachie; Goel, Aviva; Gönen, Mithat; Estrov, Zeev; Ebert, Benjamin L.; Chiosis, Gabriela; Nimer, Stephen D.; Bernstein, Bradley E.; Verstovsek, Srdan; Levine, Ross L.

2012-01-01

The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors. PMID:22820254

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

PubMed

Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

2016-12-01

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be

Structure Based Discovery of Pan Active Botulinum Neurotoxin Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vieni, Casey; McGillick, Brian; Kumaran, Desigan

Clostridium botulinum neurotoxins (BoNTs) released by the bacterium Clostridium botulinum are the most potent toxins causing the fatal disease called botulism. There are seven distinct serotypes of BoNTs (A to G) released by various strains of botulinum. They all have high sequence homology and similar three-dimensional structure. The toxicity of BoNT follows a four-step process – binding, internalization, translocation, and cleavage of its target protein, one of the three components of the SNARE complex (Soluble N-ethylmaleimde-sensitive factor attachment protein receptor) required for membrane docking and neurotransmitter release. Cleavage of one of the three proteins causes blockage of neurotransmitter release leadingmore » to flaccid paralysis. Though anyone of the above four steps could be a target for developing antidotes for botulism, the catalytic domain is the most suitable target for post exposure treatment. Of the seven serotypes BoNT/A, B, E and probably F affect humans, with BoNT/A considered to be the most potent. Development of drugs for botulism is focused on serotype specific inhibitors, but a pan-active inhibitor acting on several serotypes is preferable since it is difficult to identify the serotype before the treatment, especially since there is at least a 36-hour window before botulism can be diagnosed. Using structure-based drug discovery, we have developed three heptapeptides based on the SNARE proteins which inhibit BoNT/A, B and E equally well. Probable reasons for pan-activity of these peptides are discussed.« less

Structure Based Discovery of Pan Active Botulinum Neurotoxin Inhibitors

DOE PAGES

Vieni, Casey; McGillick, Brian; Kumaran, Desigan; ...

2018-02-14

Clostridium botulinum neurotoxins (BoNTs) released by the bacterium Clostridium botulinum are the most potent toxins causing the fatal disease called botulism. There are seven distinct serotypes of BoNTs (A to G) released by various strains of botulinum. They all have high sequence homology and similar three-dimensional structure. The toxicity of BoNT follows a four-step process – binding, internalization, translocation, and cleavage of its target protein, one of the three components of the SNARE complex (Soluble N-ethylmaleimde-sensitive factor attachment protein receptor) required for membrane docking and neurotransmitter release. Cleavage of one of the three proteins causes blockage of neurotransmitter release leadingmore » to flaccid paralysis. Though anyone of the above four steps could be a target for developing antidotes for botulism, the catalytic domain is the most suitable target for post exposure treatment. Of the seven serotypes BoNT/A, B, E and probably F affect humans, with BoNT/A considered to be the most potent. Development of drugs for botulism is focused on serotype specific inhibitors, but a pan-active inhibitor acting on several serotypes is preferable since it is difficult to identify the serotype before the treatment, especially since there is at least a 36-hour window before botulism can be diagnosed. Using structure-based drug discovery, we have developed three heptapeptides based on the SNARE proteins which inhibit BoNT/A, B and E equally well. Probable reasons for pan-activity of these peptides are discussed.« less

E7090, a Novel Selective Inhibitor of Fibroblast Growth Factor Receptors, Displays Potent Antitumor Activity and Prolongs Survival in Preclinical Models.

PubMed

Watanabe Miyano, Saori; Yamamoto, Yuji; Kodama, Kotaro; Miyajima, Yukiko; Mikamoto, Masaki; Nakagawa, Takayuki; Kuramochi, Hiroko; Funasaka, Setsuo; Nagao, Satoshi; Sugi, Naoko Hata; Okamoto, Kiyoshi; Minoshima, Yukinori; Nakatani, Yusuke; Karoji, Yuki; Ohashi, Isao; Yamane, Yoshinobu; Okada, Toshimi; Matsushima, Tomohiro; Matsui, Junji; Iwata, Masao; Uenaka, Toshimitsu; Tsuruoka, Akihiko

2016-11-01

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Relationship between the enzymatic browning and phenylalanine ammonia-lyase activity of cut lettuce, and the prevention of browning by inhibitors of polyphenol biosynthesis.

PubMed

Hisaminato, H; Murata, M; Homma, S

2001-05-01

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.

Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ladziata, Vladimir; Glunz, Peter W.; Zou, Yan

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Efficacy and safety of TNF-α inhibitors for active ankylosing spondylitis patients: Multiple treatment comparisons in a network meta-analysis

NASA Astrophysics Data System (ADS)

Liu, Wei; Wu, Yuan-Hao; Zhang, Lei; Liu, Xiao-Ya; Bin Xue; Bin Liu; Wang, Yi; Ji, Yang

2016-09-01

Ankylosing spondylitis (AS) is an inflammatory rheumatic disease with impact on axial skeleton, peripheral joints and enthuses, and it may result in severe disabilities of those parts. Tumor necrosis factor-α (TNF-α) inhibitors are considered as an effective treatment for patients with active AS. In this study, we conducted a network meta-analysis to compare the clinical outcomes of active AS patients treated with TNF-α inhibitors. Randomized controlled trials (RCTs) evaluating the efficacy and safety of TNF-α inhibitors were retrieved in literature search and selected for meta-analysis. Changes in ASAS20 response, ASAS40 response and BASDAI 50% response were regarded as efficacy outcomes; serious adverse events (SAE) and all cause withdrawals were regarded as safety outcomes. Both traditional pairwise meta-analysis and network meta-analysis were performed. The results showed that adalimumab and infliximab had better clinical outcomes. Infliximab consistently appeared to be the most effective TNF-α inhibitors with a high risk of adverse events for patients with active AS; meanwhile, adalimumab ranked highest with respect to adverse effects with efficacy secondary to infliximab. As a result, we were unable to conclude the optimal TNF-α inhibitor and this issue should be solved by future researchers.

Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor

PubMed Central

Dawson, Charlotte S.; McKenna, James A.; Quimbar, Pedro; Hayes, Brigitte M. E.; van der Weerden, Nicole L.

2017-01-01

ABSTRACT Defensins are a large family of small, cationic, cysteine-rich proteins that are part of the defense arsenal that plants use for protection against potentially damaging fungal infections. The plant defensin NaD1 from Nicotiana alata is a potent antifungal protein that inhibits growth and kills a variety of fungal pathogens that affect both plant and animal (human) hosts. Some serine protease inhibitors have also been reported to be antifungal molecules, while others have no inhibitory activity against fungi. Here we describe the synergistic activity of the plant defensin NaD1 with a selection of serine protease inhibitors against the plant pathogens Fusarium graminearum and Colletotrichum graminicola and the animal pathogen Candida albicans. The synergistic activity was not related to the protease inhibitory activity of these molecules but may arise from activation of fungal stress response pathways. The bovine pancreatic trypsin inhibitor (BPTI) displayed the most synergy with NaD1. BPTI also acted synergistically with several other antifungal molecules. The observation that NaD1 acts synergistically with protease inhibitors provides the foundation for the design of transgenic plants with improved resistance to fungal disease. It also supports the possibility of naturally occurring accessory factors that function to enhance the activity of innate immunity peptides in biological systems. IMPORTANCE This work describes the increased activity of a natural antifungal peptide in the presence of another antifungal peptide from a different family. This is termed antifungal synergy. Synergy is important for decreasing the amount of antifungal molecule needed to control the disease. Traditionally, naturally occurring antifungal molecules are assayed in isolation. Identification of synergistic interactions between antifungal peptides means that their activities in a complex biological system are likely to be different from what we observe when examining them

Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar G.; Swaminathan S.; Kumaran, D.

Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} ofmore » 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.« less

Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling.

PubMed

Kumar, Gyanendra; Kumaran, Desigan; Ahmed, S Ashraf; Swaminathan, Subramanyam

2012-05-01

Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC(50) of 0.9 µM and a K(i) of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features. © 2012 International Union of Crystallography

A new structural class of proteasome inhibitors that prevent NF-kappa B activation.

PubMed

Lum, R T; Kerwar, S S; Meyer, S M; Nelson, M G; Schow, S R; Shiffman, D; Wick, M M; Joly, A

1998-05-01

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit IC50 values ranging from 0.1 to 0.5 microgram/mL (0.1 to 1 microM). In cell proliferation assays, these compounds inhibit growth with an IC50 ranging from 5 to 10 micrograms/mL (10-20 microM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu-D-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor kappa B (NF-kappa B) in vitro by preventing signal-induced degradation of I kappa B-alpha. In these studies, the I kappa B-alpha that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit I kappa B-alpha kinase, the enzyme responsible for signal-induced phosphorylation of I kappa B-alpha. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 +/- 59 and 83 +/- 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 +/- 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-kappa B activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-kappa B.

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement.

PubMed

Zhan, Jing; Gu, Zhi-yuan; Wu, Li-qun; Zhang, Yin-kai; Hu, Ji-an

2005-06-20

The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Discovery and evaluation of inhibitors to the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1): Probing the active site-inhibitor interactions.

PubMed

Tomek, Petr; Palmer, Brian D; Flanagan, Jack U; Sun, Chuanwen; Raven, Emma L; Ching, Lai-Ming

2017-01-27

High expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) for a broad range of malignancies is associated with poor patient prognosis, and the enzyme is a validated target for cancer intervention. To identify novel IDO1 inhibitors suitable for drug development, 1597 compounds in the National Cancer Institute Diversity Set III library were tested for inhibitory activity against recombinant human IDO1. We retrieved 35 hits that inhibited IDO1 activity >50% at 20 μM. Five structural filters and the PubChem Bioassay database were used to guide the selection of five inhibitors with IC 50 between 3 and 12 μM for subsequent experimental evaluation. A pyrimidinone scaffold emerged as being the most promising. It showed excellent cell penetration, negligible cytotoxicity and passed four out of the five structural filters applied. To evaluate the importance of Ser167 and Cys129 residues in the IDO1 active site for inhibitor binding, the entire NCI library was subsequently screened against alanine-replacement mutant enzymes of these two residues. The results established that Ser167 but not Cys129 is important for inhibitory activity of a broad range of IDO1 inhibitors. Structure-activity-relationship studies proposed substituents interacting with Ser167 on four investigated IDO1 inhibitors. Three of these four Ser167 interactions associated with an increased IDO1 inhibition and were correctly predicted by molecular docking supporting Ser167 as an important mediator of potency for IDO1 inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

PubMed Central

Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas

2016-01-01

JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457

Health economics of treating haemophilia A with inhibitors.

PubMed

Knight, C

2005-11-01

Haemophilia is a rare, inherited blood disorder in which blood clotting is impaired such that patients suffer from excessive internal and external bleeding. At present there is no cure for haemophilia A and patients require expensive, life-long treatment involving clotting factor replacement therapy. Treatment costs are perceived to be higher for patients who have developed inhibitory antibodies to factor VIII, the standard therapy for haemophilia A. However, initial cost analyses suggest that clotting factor therapy with alternative haemostatic agents, such as recombinant activated factor VII or activated prothrombin complex concentrate, is no more expensive for the majority of haemophilia A patients with inhibitors than for those without inhibitors. With the availability of effective alternative haemostatic agents, orthopaedic surgery for haemophilia A patients with inhibitors is now a clinical option, and initial cost analyses suggest this may be a cost-effective treatment strategy for patients with inhibitors whose quality of life (QoL) is severely impaired by joint arthropathy. In an era of finite healthcare resourcing it is important to determine whether new treatments justify higher unit costs compared with standard therapies and whether such higher costs are justified from an individual perspective in terms of improved QoL, and from a societal perspective in terms of improved productivity and reduced overall healthcare costs. This paper examines current data on the health economics of treating haemophilia A patients with inhibitors, focusing on the overall costs of clotting factor replacement therapy and the cost consequences of joint replacement.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

PubMed

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

PubMed Central

Illendula, Anuradha; Pulikkan, John A.; Zong, Hongliang; Grembecka, Jolanta; Xue, Liting; Sen, Siddhartha; Zhou, Yunpeng; Boulton, Adam; Kuntimaddi, Aravinda; Gao, Yan; Rajewski, Roger A.; Guzman, Monica L.; Castilla, Lucio H.; Bushweller, John H.

2015-01-01

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers. PMID:25678665

[Insect cholinesterases and irreversible inhibitors. Statistical treatment of the data].

PubMed

Moralev, S N

2010-01-01

The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.

α-Amylase inhibitor activity of endophytic bacteria isolated from Annona muricata L

NASA Astrophysics Data System (ADS)

Pujiyanto, Sri; Resdiani, Merysa; Raharja, Budi; Siti Ferniah, Rejeki

2018-05-01

α-amylase (α-1,4-glucan-4-glucohydrolase, EC 3.2.1.1) is an enzyme that catalyzes the degradation of starch into its monomers. Most people use medicinal plants for keeping normal level of blood glucose, for example, the Annona muricata. The objectives of this study are to obtain endophytic bacteria from the plant, knowing the activity of the α-amylase inhibitor of selected isolates. Endophytic bacteria are isolated from the roots, stems, and leaves of the plant have been sterilized surface and grown in NA medium. A total of 11 isolates were found to produce α-amylase inhibitor compounds. The isolates obtained were tested for their α-amylase inhibitor activity, and isolates with the highest activity tested further. Isolate DS21 show the best activity with 72,22% inhibition. The experimental design used in this research is Completely Randomized Design (RAL). The best isolates treated by a variety of carbon sources, and the best carbon source treated with various pH. The data obtained were analyzed usingAnalysis of Variance (ANOVA). The results of statistical tests show the treatment of starch and lactose has a significant effect on the production of α-amylase inhibitors (P <0.05) and the pH 5 and 6,0 significantly affected the production of α-amylase inhibitors (P <0.05).

Discovery of novel 4-anilinoquinazoline derivatives as potent inhibitors of epidermal growth factor receptor with antitumor activity.

PubMed

Xu, Yun-Yun; Li, Si-Ning; Yu, Gao-Jian; Hu, Qing-Hua; Li, Huan-Qiu

2013-10-01

Two new series of new compounds containing a 6-amino-substituted group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus have been discovered as potential EGFR inhibitors. These compounds proved efficient effects on antiproliferative activity and EGFR-TK inhibitory activity. Especially, N(6)-((5-bromothiophen-2-yl)methyl)-N(4)-(3-chlorophenyl)quinazoline-4,6-diamine (5e), showed the most potent inhibitory activity (IC50=3.11μM for Hep G2, IC50=0.82μM for A549). The EGFR molecular docking model suggested that the new compound is nicely bound to the region of EGFR, and cell morphology by Hoechst stain experiment suggested that these compounds efficiently induced apoptosis of A549 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

PubMed

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-05-10

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor

PubMed Central

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-01-01

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted. PMID:25857298

Risk Factors for Inhibitor Formation in Hemophilia: A Prevalent Case-Control Study

PubMed Central

Ragni, Margaret V.; Ojeifo, Oluseyi; Feng, Jinong; Yan, Jin; Hill, Kathleen A.; Sommer, Steve S.; Trucco, Massimo N.; Brambilla, Donald J.

2009-01-01

Background Inhibitor formation is a major complication of hemophilia treatment. Aim In a prevalent case-control study, we evaluated blood product exposure, genotype, and HLA type on hemophilia A inhibitor formation. Methods Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Results Cases experienced higher intensity factor, 455 vs. 200 U per exposure, p<0.005, more frequent central nervous system (CNS) bleeding, 7 of 20 (35.0%) vs. 1 of 57 (1.7%), p=0.001, and more commonly from inhibitor families, 7 of 20 (35.0%) vs. 0 of 57 (0%), p<0.001, and African-American, 12 of 63 (19.0%) vs. 6 of 117 (5.1%), p=0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, p<0.001, and survival was shorter, 14 vs. 38 yr, p=0.025. Inhibitor formation was uncommon in those with missense mutations, 2 of 65 (3.1%) vs. 31 of 119 (26.0%), p=0.008, and unrelated to factor VIII immunogenic epitope, p=0.388, or HLA type, p>0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titers < 120 vs. ≥ 120 BU/ml, 6 vs. 16 months, p<0.01, but unaffected by tolerizing dose regimen, p>0.50. Conclusions Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race, and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies. PMID:19563499

Antimalarial activity of HIV-1 protease inhibitor in chromone series.

PubMed

Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

2014-12-01

Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis

PubMed Central

Bharat, Amrita; Blanchard, Jan E.; Brown, Eric D.

2014-01-01

The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis. PMID:23606650

Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

PubMed

Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

2013-11-01

Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A

The IGF-1 receptor inhibitor picropodophyllin potentiates the anti-myeloma activity of a BH3-mimetic

PubMed Central

Bieghs, Liesbeth; Lub, Susanne; Fostier, Karel; Maes, Ken; Van Valckenborgh, Els; Menu, Eline; Johnsen, Hans E.; Overgaard, Michael T.; Larsson, Olle; Axelson, Magnus; Nyegaard, Mette; Schots, Rik; Jernberg-Wiklund, Helena

2014-01-01

The ABT-analogous 737, 263 and 199 are BH3 mimetics showing potent anti-myeloma (MM) activity, but only on defined molecular subgroups of MM patients presenting a Bcl-2high/Mcl-1low profile. IGF-1 is a major survival factor in MM regulating the expression of Bcl-2 proteins and might therefore be a resistance factor to these ABT-analogous. We first show that IGF-1 protected human MM cell lines (HMCLs) against ABT-737. Concurrently, the IGF-1 receptor inhibitor picropodophyllin (PPP) synergistically sensitized HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Knockdown of Bcl-2 by shRNA protected MM cells to ABT-737, while Mcl-1 shRNA sensitized the cells. PPP overcame the Bcl-2 dependency of ABT-737, but failed to completely overcome the protective effect of Mcl-1. In vivo, co-treatment of 5T33MM bearing mice significantly decreased tumor burden and prolonged overall survival both in a prophylactic and therapeutic setting. Interestingly, proteasome inhibitor resistant CD138− 5T33MM cells were more sensitive to ABT-737, whereas PPP alone targeted the CD138+ cells more effectively. After co-treatment, both subpopulations were targeted equally. Together, the combination of an IGF-1R inhibitor and an ABT-analogue displays synergistic anti-myeloma activity providing the rational for further (pre)clinical testing. PMID:25008202

QSAR and 3D QSAR of inhibitors of the epidermal growth factor receptor

NASA Astrophysics Data System (ADS)

Pinto-Bazurco, Mariano; Tsakovska, Ivanka; Pajeva, Ilza

This article reports quantitative structure-activity relationships (QSAR) and 3D QSAR models of 134 structurally diverse inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. Free-Wilson analysis was used to derive the QSAR model. It identified the substituents in aniline, the polycyclic system, and the substituents at the 6- and 7-positions of the polycyclic system as the most important structural features. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used in the 3D QSAR modeling. The steric and electrostatic interactions proved the most important for the inhibitory effect. Both QSAR and 3D QSAR models led to consistent results. On the basis of the statistically significant models, new structures were proposed and their inhibitory activities were predicted.

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074.

PubMed

Sanfelice, Domenico; Koss, Hans; Bunney, Tom D; Thompson, Gary S; Farrell, Brendan; Katan, Matilda; Breeze, Alexander L

2018-03-26

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

LPS-inducible factor(s) from activated macrophages mediates cytolysis of Naegleria fowleri amoebae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cleary, S.F.; Marciano-Cabral, F.

1986-03-01

Soluble cytolytic factors of macrophage origin have previously been described with respect to their tumoricidal activity. The purpose of this study was to investigate the mechanism and possible factor(s) responsible for cytolysis of the amoeba Naegleria fowleri by activated peritoneal macrophages from B6C3F1 mice. Macrophages or conditioned medium (CM) from macrophage cultures were incubated with /sup 3/H-Uridine labeled amoebae. Percent specific release of label served as an index of cytolysis. Bacille Calmette-Guerin (BCG) and Corynebacterium parvum macrophages demonstrated significant cytolysis of amoebae at 24 h with an effector to target ratio of 10:1. Treatment of macrophages with inhibitors of RNAmore » or protein synthesis blocked amoebicidal activity. Interposition of a 1 ..mu..m pore membrane between macrophages and amoebae inhibited killing. Inhibition in the presence of the membrane was overcome by stimulating the macrophages with LPS. CM from SPS-stimulated, but not unstimulated, cultures of activated macrophages was cytotoxic for amoebae. The activity was heat sensitive and was recovered from ammonium sulfate precipitation of the CM. Results indicate that amoebicidal activity is mediated by a protein(s) of macrophage origin induced by target cell contact or stimulation with LPS.« less

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines

PubMed Central

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-01-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

PubMed

Samac, D; Storey, R

1981-12-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

Effect of wine inhibitors on free pineapple stem bromelain activity in a model wine system.

PubMed

Esti, Marco; Benucci, Ilaria; Liburdi, Katia; Garzillo, Anna Maria Vittoria

2011-04-13

The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.

Simm530, a novel and highly selective c-Met inhibitor, blocks c-Met-stimulated signaling and neoplastic activities

PubMed Central

Peng, Xia; Shen, Yanyan; Chen, Fang; Ji, Yinchun; Liu, Weiren; Shi, Yinghong; Duan, Wenhu; Ding, Jian; Ai, Jing; Geng, Meiyu

2016-01-01

The aberrant c-Met activation has been implicated in a variety of human cancers for its critical role in tumor growth, metastasis and tumor angiogenesis. Thus, c-Met axis presents as an attractive therapeutic target. Notably, most of these c-Met inhibitors currently being evaluated in clinical trials lack selectivity and target multiple kinases, often accounting for the undesirable toxicities. Here we described Simm530 as a potent and selective c-Met inhibitor. Simm530 demonstrated >2,000 fold selectivity for c-Met compared with other 282 kinases, making it one of the most selective c-Met inhibitors described to date. This inhibitor significantly blocked c-Met signaling pathways regardless of mechanistic complexity implicated in c-Met activation. As a result, Simm530 led to substantial inhibition of c-Met-promoted cell proliferation, migration, invasion, ECM degradation, cell scattering and invasive growth. In addition, Simm530 inhibited primary human umbilical vascular endothelial cell (HUVEC) proliferation, decreased intratumoral CD31 expression and plasma pro-angiogenic factor interleukin-8 secretion, suggesting its significant anti-angiogenic properties. Simm530 resulted in dose-dependent inhibition of c-Met phosphorylation and tumor growth in c-Met-driven lung and gastric cancer xenografts. And, the inhibitor is well tolerated even at doses that achieve complete tumor regression. Together, Simm530 is a potent and highly selective c-Met kinase inhibitor that may have promising therapeutic potential in c-Met-driven cancer treatment. PMID:27191264

COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

PubMed Central

Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

2013-01-01

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakatani, Miyuki; Ito, Jumpei; Japan Society for the Promotion of Science, Tokyo, 102-0083

Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the firstmore » step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.« less

Iminopyrimidinones: A novel pharmacophore for the development of orally active renin inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKittrick, Brian A.; Caldwell, John P.; Bara, Thomas

2015-04-01

The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

Oximes: Inhibitors of Human Recombinant Acetylcholinesterase. A Structure-Activity Relationship (SAR) Study

PubMed Central

Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J.; Kuca, Kamil

2013-01-01

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring. PMID:23959117

Role of Epidermal Growth Factor Receptor (EGFR) Inhibitors and Radiation in the Management of Brain Metastases from EGFR Mutant Lung Cancers.

PubMed

Khandekar, Melin J; Piotrowska, Zofia; Willers, Henning; Sequist, Lecia V

2018-04-27

The growth of genotype-directed targeted therapies, such as inhibitors of the epidermal growth factor receptor (EGFR), has revolutionized treatment for some patients with oncogene-addicted lung cancer. However, as systemic control for these patients has improved, brain metastases remain an important source of morbidity and mortality. Traditional treatment for brain metastases has been radiotherapy, either whole-brain radiation or stereotactic radiosurgery. The growing availability of drugs that can cross the blood-brain barrier and have activity in the central nervous system (CNS) has led to many studies investigating whether targeted therapy can be used in combination with or in lieu of radiation. In this review, we summarize the key literature about the incidence and nature of EGFR-mutant brain metastases (EGFR BMs), the data about the activity of EGFR inhibitors in the CNS, and whether they can be used as front-line therapy for brain metastases. Although initial use of tyrosine kinase inhibitors for EGFR BMs can often be an effective treatment strategy, multidisciplinary evaluation is critical, and prospective studies are needed to clarify which patients may benefit from early radiotherapy. Management of brain metastases in epidermal growth factor receptor (EGFR) mutant lung cancer is a common clinical problem. The question of whether to start initial therapy with an EGFR inhibitor or radiotherapy (either whole-brain radiotherapy or stereotactic radiosurgery) is controversial. The development of novel EGFR inhibitors with enhanced central nervous system (CNS) penetration is an important advance in the treatment of CNS disease. Multidisciplinary evaluation and evaluation of extracranial disease status are critical to choosing the best treatment option for each patient. © AlphaMed Press 2018.

Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation*

PubMed Central

Alvarez-Flores, Miryam Paola; Furlin, Daniel; Ramos, Oscar H. P.; Balan, Andrea; Konno, Katsuhiro; Chudzinski-Tavassi, Ana Marisa

2011-01-01

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X. PMID:21177860

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

Discovery of Selective and Noncovalent Diaminopyrimidine-Based Inhibitors of Epidermal Growth Factor Receptor Containing the T790M Resistance Mutation

DOE PAGES

Hanan, Emily J.; Eigenbrot, Charles; Bryan, Marian C.; ...

2014-11-10

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. Here in this paper, wemore » 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.« less

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501

2008-04-01

The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less

Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

PubMed

Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R

2007-07-01

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

The SIRT1 inhibitor EX-527 suppresses mTOR activation and alleviates acute lung injury in mice with endotoxiemia.

PubMed

Huang, Jing; Tian, Rui; Yang, Yongqiang; Jiang, Rong; Dai, Jie; Tang, Li; Zhang, Li

2017-11-01

It is generally regarded that Sirtuin 1 (SIRT1), a longevity factor in mammals, acts as a negative regulator of inflammation. However, recent studies also found that SIRT1 might be a detrimental factor under certain inflammatory circumstance. In this study, the potential pathophysiological roles and the underlying mechanisms of SIRT1 in a mouse model with endotoxemia-associated acute lung injury were investigated. The results indicated that treatment with the selective SIRT1 inhibitor EX-527 suppressed LPS-induced elevation of TNF-α and IL-6 in plasma. Treatment with EX-527 attenuated LPS-induced histological abnormalities in lung tissue, which was accompanied with decreased myeloperoxidase level and suppressed induction of tissue factor and plasminogen activator inhibitor-1. Treatment with EX-527 also suppressed LPS-induced phosphorylation of eukaryotic translation initiation factor-binding protein 1 (4E-BP1). Co-administration of a mammalian target of rapamycin (mTOR) activator 3-benzyl-5-[(2-nitrophenoxy) methyl]-dihydrofuran-2 (3H)-one (3BDO) abolished the inhibitory effects of EX-527 on 4E-BP1 phosphorylation. Meanwhile, the inhibitory effects of EX-527 on IL-6 induction and the beneficial effects of EX-527 on lung injury were partially reversed by 3BDO. This study suggests that selective inhibition of SIRT1 by EX-527 might alleviate endotoxemia-associated acute lung injury partially via suppression of mTOR, which implies that SIRT1 selective inhibitors might have potential value for the pharmacological intervention of inflammatory lung injury.

Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein Inhibitors Involve Redox-Sensitive and Signal Transducer and Activator of Transcription 3-Dependent Pathways.

PubMed

Rios, Francisco J; Lopes, Rheure A; Neves, Karla B; Camargo, Livia L; Montezano, Augusto C; Touyz, Rhian M

2016-05-01

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which

Initial Assessment, Surveillance, and Management of Blood Pressure in Patients Receiving Vascular Endothelial Growth Factor Signaling Pathway Inhibitors

PubMed Central

Bakris, George L.; Black, Henry R.; Chen, Helen X.; Durand, Jean-Bernard; Elliott, William J.; Ivy, S. Percy; Leier, Carl V.; Lindenfeld, JoAnn; Liu, Glenn; Remick, Scot C.; Steingart, Richard; Tang, W. H. Wilson

2010-01-01

Hypertension is a mechanism-based toxic effect of drugs that inhibit the vascular endothelial growth factor signaling pathway (VSP). Substantial evidence exists for managing hypertension as a chronic condition, but there are few prospectively collected data on managing acute hypertension caused by VSP inhibitors. The Investigational Drug Steering Committee of the National Cancer Institute convened an interdisciplinary cardiovascular toxicities expert panel to evaluate this problem, to make recommendations to the Cancer Therapy Evaluation Program on further study, and to structure an approach for safe management by treating physicians. The panel reviewed: the published literature on blood pressure (BP), hypertension, and specific VSP inhibitors; abstracts from major meetings; shared experience with the development of VSP inhibitors; and established principles of hypertension care. The panel generated a consensus report including the recommendations on clinical concerns summarized here. To support the greatest possible number of patients to receive VSP inhibitors safely and effectively, the panel had four recommendations: 1) conduct and document a formal risk assessment for potential cardiovascular complications, 2) recognize that preexisting hypertension will be common in cancer patients and should be identified and addressed before initiation of VSP inhibitor therapy, 3) actively monitor BP throughout treatment with more frequent assessments during the first cycle of treatment, and 4) manage BP with a goal of less than 140/90 mmHg for most patients (and to lower, prespecified goals in patients with specific preexisting cardiovascular risk factors). Proper agent selection, dosing, and scheduling of follow-up should enable maintaining VSP inhibition while avoiding the complications associated with excessive or prolonged elevation in BP. PMID:20351338

Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

PubMed

Arnadottir, M; Nilsson-Ehle, P

1994-01-01

The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

RecA Inhibitors Potentiate Antibiotic Activity and Block Evolution of Antibiotic Resistance.

PubMed

Alam, Md Kausar; Alhhazmi, Areej; DeCoteau, John F; Luo, Yu; Geyer, C Ronald

2016-03-17

Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, β-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life. Copyright © 2016 Elsevier Ltd. All rights reserved.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

Mechanism of action of recombinant activated factor VII: an update.

PubMed

Hedner, Ulla

2006-01-01

Bleeding episodes in patients with hemophilia and inhibitors must be managed using agents that are hemostatically active in the absence of factor VIII or IX. Activated prothrombin complex concentrates have long been used in this context. However, the search for safer and more effective agents has led to the development of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark). This paper presents an update on the mechanism of action of rFVIIa, and describes how pharmacologic doses of this agent enhance thrombin production and thus contribute to the development of a stable, lysis-resistant fibrin plug at the site of vessel damage. This mechanism explains the reported efficacy of rFVIIa in a range of clinical situations characterized by impaired thrombin generation.

Antithrombotic effects of factor Xa inhibition with DU-176b: Phase-I study of an oral, direct factor Xa inhibitor using an ex-vivo flow chamber.

PubMed

Zafar, Mohammad Urooj; Vorchheimer, David A; Gaztanaga, Juan; Velez, Mauricio; Yadegar, Daniel; Moreno, Pedro R; Kunitada, Satoshi; Pagan, Juan; Fuster, Valentin; Badimon, Juan J

2007-10-01

Direct and specific inhibition of factor Xa is an emerging therapeutic strategy for atherothrombotic disease. Parenteral factor Xa inhibitors promise efficacy comparable to standard therapies, which could be extended to ambulatory patients with oral agents. We evaluated the antithrombotic effect of the oral, direct factor Xa inhibitor DU-176b in a phase-I study. Healthy subjects (n = 12) received a single, 60 mg dose of DU-176b. Antithrombotic effects were assessed by comparing ex-vivo thrombus formation at 1.5, 5, and 12 hours post-dose versus baseline, along with factor Xa activity, thrombin generation and clotting parameters. Under venous flow after 1.5 and 5 hours, the thrombus was 28% and 21% smaller versus baseline, respectively (p < 0.05). Under arterial condition, the reduction was 26% and 17% (p < 0.05). Thrombin generation decreased by 28% at 1.5 hours and 10% at 5 hours. Changes in PT and INR correlated well with plasma drug concentrations (R2 = 0.79 and 0.78). Direct and specific inhibition of factor Xa by DU-176b significantly reduced ex-vivo thrombus formation at both venous and arterial rheologies, up to 5 hours post-dose. The effects mirrored changes in clotting parameters, suggesting their potential usefulness for monitoring in a clinical setting.

Identification and characterisation of carnostatine (SAN9812), a potent and selective carnosinase (CN1) inhibitor with in vivo activity.

PubMed

Qiu, Jiedong; Hauske, Sibylle J; Zhang, Shiqi; Rodriguez-Niño, Angelica; Albrecht, Thomas; Pastene, Diego O; van den Born, Jacob; van Goor, Harry; Ruf, Sven; Kohlmann, Markus; Teufel, Michael; Krämer, Bernhard K; Hammes, Hans-Peter; Peters, Verena; Yard, Benito A; Kannt, Aimo

2018-06-20

Carnosinase 1 (CN1) has been postulated to be a susceptibility factor for developing diabetic nephropathy (DN). Although its major substrate, carnosine, is beneficial in rodent models of DN, translation of these findings to humans has been hampered by high CN1 activity in human serum resulting in rapid degradation of carnosine. To overcome this hurdle, we screened a protease-directed small-molecule library for inhibitors of human recombinant CN1. We identified SAN9812 as a potent and highly selective inhibitor of CN1 activity with a K i of 11 nM. It also inhibited CN1 activity in human serum and serum of transgenic mice-overexpressing human CN1. Subcutaneous administration of 30 mg/kg SAN9812 led to a sustained reduction in circulating CN1 activity in human CN1 transgenic (TG) mice. Simultaneous administration of carnosine and SAN9812 increased carnosine levels in plasma and kidney by up to 100-fold compared to treatment-naïve CN1-overexpressing mice. To our knowledge, this is the first study reporting on a potent and selective CN1 inhibitor with in vivo activity. SAN9812, also called carnostatine, may be used to increase renal carnosine concentration as a potential therapeutic modality for renal diseases linked to glycoxidative conditions.

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

PubMed

Krajewska, Barbara; Zaborska, Wiesława

2007-10-01

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

PubMed Central

Proia, David A.; Foley, Kevin P.; Korbut, Tim; Sang, Jim; Smith, Don; Bates, Richard C.; Liu, Yuan; Rosenberg, Alex F.; Zhou, Dan; Koya, Keizo; Barsoum, James; Blackman, Ronald K.

2011-01-01

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2V617F mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors. PMID:21533169

Recombinant activated factor VII: 30 years of research and innovation.

PubMed

Hedner, Ulla

2015-06-01

Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Incensole acetate, a novel anti-inflammatory compound isolated from Boswellia resin, inhibits nuclear factor-kappa B activation.

PubMed

Moussaieff, Arieh; Shohami, Esther; Kashman, Yoel; Fride, Ester; Schmitz, M Lienhard; Renner, Florian; Fiebich, Bernd L; Munoz, Eduardo; Ben-Neriah, Yinon; Mechoulam, Raphael

2007-12-01

Boswellia resin is a major anti-inflammatory agent in herbal medical tradition, as well as a common food supplement. Its anti-inflammatory activity has been attributed to boswellic acid and its derivatives. Here, we re-examined the anti-inflammatory effect of the resin, using inhibitor of nuclear factor-kappaB alpha (IkappaB alpha) degradation in tumor necrosis factor (TNF) alpha-stimulated HeLa cells for a bioassay-guided fractionation. We thus isolated two novel nuclear factor-kappaB (NF-kappaB) inhibitors from the resin, their structures elucidated as incensole acetate (IA) and its nonacetylated form, incensole (IN). IA inhibited TAK/TAB-mediated IkappaB kinase (IKK) activation loop phosphorylation, resulting in the inhibition of cytokine and lipopolysaccharide-mediated NF-kappaB activation. It had no effect on IKK activity in vitro, and it did not suppress IkappaB alpha phosphorylation in costimulated T-cells, indicating that the kinase inhibition is neither direct nor does it affect all NF-kappaB activation pathways. The inhibitory effect seems specific; IA did not interfere with TNFalpha-induced activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. IA treatment had a robust anti-inflammatory effect in a mouse inflamed paw model. Cembrenoid diterpenoids, specifically IA and its derivatives, may thus constitute a potential novel group of NF-kappaB inhibitors, originating from an ancient anti-inflammatory herbal remedy.

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…

Hit identification of IKKβ natural product inhibitor.

PubMed

Leung, Chung-Hang; Chan, Daniel Shiu-Hin; Li, Ying-Wei; Fong, Wang-Fun; Ma, Dik-Lung

2013-01-07

The nuclear factor-κB (NF-κB) proteins are a small group of heterodimeric transcription factors that play an important role in regulating the inflammatory, immune, and apoptotic responses. NF-κB activity is suppressed by association with the inhibitor IκB. Aberrant NF-κB signaling activity has been associated with the development of cancer, chronic inflammatory diseases and auto-immune diseases. The IKK protein complex is comprised of IKKα, IKKβ and NEMO subunits, with IKKβ thought to play the dominant role in modulating NF-κB activity. Therefore, the discovery of new IKKβ inhibitors may offer new therapeutic options for the treatment of cancer and inflammatory diseases. A structure-based molecular docking approach has been employed to discover novel IKKβ inhibitors from a natural product library of over 90,000 compounds. Preliminary screening of the 12 highest-scoring compounds using a luciferase reporter assay identified 4 promising candidates for further biological study. Among these, the benzoic acid derivative (1) showed the most promising activity at inhibiting IKKβ phosphorylation and TNF-α-induced NF-κB signaling in vitro. In this study, we have successfully identified a benzoic acid derivative (1) as a novel IKKβ inhibitor via high-throughput molecular docking. Compound 1 was able to inhibit IKKβ phosphorylation activity in vitro, and block IκBα protein degradation and subsequent NF-κB activation in human cells. Further in silico optimization of the compound is currently being conducted in order to generate more potent analogues for biological tests.

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

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

2016-10-04

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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effects of tumor necrosis factor alpha antagonist, platelet activating factor antagonist, and nitric oxide synthase inhibitor on experimental otitis media with effusion.

PubMed

Kim, Dong-Hyun; Park, Yong-Soo; Jeon, Eun-Ju; Yeo, Sang-Won; Chang, Ki-Hong; Lee, Seung Kyun

2006-08-01

We studied the inflammatory responses in otitis media with effusion induced by lipopolysaccharide (LPS) in rats, and compared the preventive effects of tumor necrosis factor (TNF) soluble receptor type I (sTNFRI, a TNF-alpha antagonist), platelet activating factor antagonist, and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). We used 2 control groups of Sprague Dawley rats (untreated and saline-treated) and 4 experimental groups, which all received an intratympanic injection of LPS, followed in 3 groups by experimental treatment of the same ear. The LPS group had no additional treatment. The L-NAME group received intraperitoneal injection of L-NAME and was reinjected after 12 hours. The A-85783 group was first given an intraperitoneal injection of A-85783. The sTNFRI group was first given an intratympanic injection of sTNFRI. Twenty-four hours after the initial intratympanic injection of LPS, temporal bones from each group were examined histopathologically and the vascular permeability of the middle ear mucosa was measured by Evans blue vital dye staining. The L-NAME, A-85783, and sTNFRI groups showed significantly reduced capillary permeability, subepithelial edema, and infiltration of inflammatory cells in comparison with the LPS group. There were no differences in capillary permeability, subepithelial edema, or infiltration of inflammatory cells between the A-85783 and sTNFRI groups. The L-NAME group showed no difference in vascular permeability or subepithelial edema in comparison with the A-85783 and sTNFRI groups, but showed more infiltration of inflammatory cells. We conclude that sTNFRI, A-85783, and L-NAME can be proposed as alternative future treatments for otitis media with effusion. However, L-NAME may be the least effective of these agents.

Mechanisms and Clinical Activity of an EGFR and HER2 Exon 20-selective Kinase Inhibitor in Non-small Cell Lung Cancer

PubMed Central

Robichaux, Jacqulyne P.; Elamin, Yasir Y.; Tan, Zhi; Carter, Brett W.; Zhang, Shuxing; Liu, Shengwu; Li, Shuai; Chen, Ting; Poteete, Alissa; Estrada-Bernal, Adriana; Le, Anh T.; Truini, Anna; Nilsson, Monique B.; Sun, Huiying; Roarty, Emily; Goldberg, Sarah B.; Brahmer, Julie R.; Altan, Mehmet; Lu, Charles; Papadimitrakopoulou, Vassiliki; Politi6, Katerina; Doebele, Robert C.; Wong, Kwok-Kin; Heymach, John V.

2018-01-01

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations. PMID:29686424

Determinants of activity of the HIV-1 maturation inhibitor PA-457.

PubMed

Li, Feng; Zoumplis, Dorian; Matallana, Claudia; Kilgore, Nicole R; Reddick, Mary; Yunus, Abdul S; Adamson, Catherine S; Salzwedel, Karl; Martin, David E; Allaway, Graham P; Freed, Eric O; Wild, Carl T

3-O-(3',3'-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

Discovery of novel aminobenzisoxazole derivatives as orally available factor IXa inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakurada, Isao; Endo, Toshiya; Hikita, Katsuyoshi

2017-06-01

Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.

Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

PubMed

Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

2016-11-01

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC 50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

PubMed

Scherrer, A; Kruithof, E K; Grob, J P

1991-06-01

Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

The strange connection between epidermal growth factor receptor tyrosine kinase inhibitors and dapsone: from rash mitigation to the increase in anti-tumor activity.

PubMed

Boccellino, Mariarosaria; Quagliuolo, Lucio; Alaia, Concetta; Grimaldi, Anna; Addeo, Raffaele; Nicoletti, Giovanni Francesco; Kast, Richard Eric; Caraglia, Michele

2016-11-01

The presence of an aberrantly activated epidermal growth factor receptor (EGFR) in many epithelial tumors, due to its overexpression, activating mutations, gene amplification and/or overexpression of receptor ligands, represent the fundamental basis underlying the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Drugs inhibiting the EGFR have different mechanisms of action; while erlotinib and gefitinib inhibit the intracellular tyrosine kinase, monoclonal antibodies like cetuximab and panitumumab bind the extracellular domain of the EGFR both activating immunomediated anti-cancer effect and inhibiting receptor function. On the other hand, interleukin-8 has tumor promoting as well as neo-angiogenesis enhancing effects and several attempts have been made to inhibit its activity. One of these is based on the use of the old sulfone antibiotic dapsone that has demonstrated several interleukin-8 system inhibiting actions. Erlotinib typically gives a rash that has recently been proven to come out via up-regulated keratinocyte interleukin-8 synthesis with histological features reminiscent of typical neutrophilic dermatoses. In this review, we report experimental evidence that shows the use of dapsone to improve quality of life in erlotinib-treated patients by ameliorating rash as well as short-circuiting a growth-enhancing aspect of erlotinib based on increased interleukin-8 secretion.

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

PubMed Central

Samac, Deborah; Storey, Richard

1981-01-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

Unraveling the Pivotal Role of Bradykinin in ACE Inhibitor Activity.

PubMed

Taddei, Stefano; Bortolotto, L

2016-10-01

Historically, the first described effect of an angiotensin converting enzyme (ACE) inhibitor was an increased activity of bradykinin, one of the substrates of ACE. However, in the subsequent years, molecular models describing the mechanism of action of ACE inhibitors in decreasing blood pressure and cardiovascular risk have focused mostly on the renin-angiotensin system. Nonetheless, over the last 20 years, the importance of bradykinin in regulating vasodilation, natriuresis, oxidative stress, fibrinolysis, inflammation, and apoptosis has become clearer. The affinity of ACE appears to be higher for bradykinin than for angiotensin I, thereby suggesting that ACE inhibitors may be more effective inhibitors of bradykinin degradation than of angiotensin II production. Data describing the effect of ACE inhibition on bradykinin signaling support the hypothesis that the most cardioprotective benefits attributed to ACE inhibition may be due to increased bradykinin signaling rather than to decreased angiotensin II signaling, especially when high dosages of ACE inhibitors are considered. In particular, modulation of bradykinin in the endothelium appears to be a major target of ACE inhibition. These new mechanistic concepts may lead to further development of strategies enhancing the bradykinin signaling.

Chitin synthesis inhibitors promote liver cancer cell metastasis via interfering with hypoxia-inducible factor 1α.

PubMed

Ning, Xia; Wang, Yue; Yan, Wei; Li, Guangke; Sang, Nan

2018-05-03

Chitin synthesis inhibitors (CSIs), as alternatives to conventional insecticides, have been in worldwide demand in recent years. However, little attention has been paid to the potential ecological safety and health risks of CSIs, especially their abilities to interfere with nonsexual hormone receptors such as hypoxia-inducible factor 1α (HIF-1α). In this work, we conducted a systematic study regarding the influence of CSIs on HIF-1α-related liver cancer cell metastasis. The dual-luciferase reporter gene assay revealed that two of fourteen CSIs exhibited dose-response HIF-1α agonistic activities at noncytotoxic concentrations with relative luciferase activity (RLA) values of 25.6% for diflubenzuron (DFB) and 20.9% for triflumuron (TFM). Following this result, in vitro bioassays demonstrated that both DFB and TFM stimulated HepG2 cell migration and invasion. This action was associated with the varied expression levels of genes involved in epithelial-to-mesenchymal transition (EMT) activation and extracellular matrix (ECM) degradation, such as the upregulation of fibronectin (FN1) and matrix metalloproteinase-2 (MMP-2) and the suppression of E-cadherin (E-cad) and tissue inhibitor of metalloproteinases-2 (TIMP-2). Moreover, changes in these EMT and ECM phenotype markers were dramatically blocked by a HIF-1α inhibitor (KC7F2), which further verified the involvement of HIF-1α in CSI-induced HepG2 cell metastasis. For the first time, our findings reveal that CSIs play crucial roles in promoting the metastasis of human liver cancer cells and that HIF-1α is potentially responsible for these changes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hypofibrinolytic state in HIV-1-infected patients treated with protease inhibitor-containing highly active antiretroviral therapy.

PubMed

Koppel, Kristina; Bratt, Göran; Schulman, Sam; Bylund, Håkan; Sandström, Eric

2002-04-15

Decreased insulin sensitivity, hyperlipidemia, and body fat changes are considered as risk factors for coronary heart disease (CHD). A clustering of such factors (metabolic syndrome [MSDR]) exponentially increases the risk. Impaired fibrinolysis and increased coagulation are additional independent risk factors for CHD. We studied the effects of protease inhibitor (PI)-containing highly active antiretroviral therapy (HAART) on metabolic and hemostatic parameters in 363 HIV-infected individuals, of whom 266 were receiving PI-containing HAART and 97 were treatment naive. The fasting plasma levels of insulin, glucose, triglycerides, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, plasminogen activator inhibitor type 1 (PAI-1), and fibrinogen were evaluated together with the areas of visceral adipose tissue and the visceral adipose tissue/subcutaneous adipose tissue area ratio. The levels of insulin, triglycerides, cholesterol, and low-density lipoprotein cholesterol; visceral adipose tissue area; low-density lipoprotein/high-density lipoprotein ratio; and visceral adipose tissue/subcutaneous adipose tissue area ratio were significantly increased in patients receiving PI-containing HAART compared with treatment-naive patients. The levels of PAI-1 and fibrinogen were significantly higher in patients receiving PI-containing HAART. PAI-1 levels were higher in individuals with MSDR but also in patients without MSDR who were receiving PI-containing HAART. PAI-1 was independently correlated to use of PI-containing HAART, triglyceride level, insulin level, and body mass index (p <.001). These findings suggest that patients receiving PI-containing HAART have decreased fibrinolysis and increased coagulability, which may thus represent additional risk factors for cardiovascular disease in this patient group.

Cost analysis of prophylaxis with activated prothrombin complex concentrate vs. on-demand therapy with activated factor VII in severe haemophilia A patients with inhibitors, in Spain.

PubMed

Villarrubia, R; Oyagüez, I; Álvarez-Román, M T; Mingot-Castellano, M E; Parra, R; Casado, M A

2015-05-01

A cost analysis model was developed to compare annual cost of prophylaxis with activated prothrombin complex concentrate (aPCC) vs. on-demand therapy with activated recombinant factor VII (rFVIIa) in severe haemophilia A patients with inhibitors for the Spanish National Health System (NHS). Model inputs were drug cost for prophylaxis (aPCC) and for on-demand treatment (rFVIIa or aPCC); bleeding episodes management (excluding bypassing agent cost); surgical costs and disease management (excluding bleeding episodes). Annual bleeding episodes treated on-demand was assumed to be 25, whereas breakthrough bleeds on prophylaxis was 8. Dose for prophylaxis was 75.72 U kg(-1) , three times per week. The total on-demand dose/bleeding episode was 679.66 μg kg(-1) (rFVIIa) and 235.28 U kg(-1) (aPCC). The average bleeding cost (€2998) considered different bleeding sites (62.5% joints, 28.6% muscles and soft tissues, 3.6% mucocutaneous tissues and 5.4% other areas). A 7.5% deduction was applied to ex-factory drug prices. Unitary costs (€2013) derived from local databases. Sensitivity analyses (SA) were performed. Annual cost of aPCC prophylaxis (€524,358) was 16% lower than on-demand treatment with rFVIIa (€627,876). Yearly drug costs were €497,017 for aPCC (€73,166 for on-demand treatment and €423,850 for prophylaxis), and €548,870 for rFVIIa. Disease management cost (€2645 per year) and surgical procedures (€708 per year) were common for both strategies. In the SA prophylactic treatment led to savings between €26,225 and €-1,008,960. Prophylaxis with aPCC reduces number of bleeding episodes in severe haemophilia A patients with inhibitors. aPCC prophylaxis resulted in savings in excess of €100,000 per-patient per year, being 16% less costly than on-demand treatment with rFVIIa, for the Spanish NHS. © 2015 John Wiley & Sons Ltd.

Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

PubMed

Stokol, Tracy; Serpa, Priscila Beatriz da Silva; Zahid, Muhammad N; Brooks, Marjory B

2016-01-01

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo . We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1-0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Cost-effectiveness of recombinant activated factor VII vs. plasma-derived activated prothrombin complex concentrate in the treatment of mild-to-moderate bleeding episodes in patients with severe haemophilia A and inhibitors in Spain.

PubMed

Jimenez-Yuste, V; Núñez, R; Romero, J A; Montoro, B; Espinós, B

2013-11-01

Several analyses have shown that recombinant activated factor VII (rFVIIa) is a cost-effective intervention compared with plasma-derived activated prothrombin complex concentrate (pd-aPCC) for the on-demand treatment of mild-to-moderate bleeds in haemophilia patients with inhibitors. The aim of the study was to assess the cost-effectiveness of rFVIIa vs. pd-aPCC in the treatment of bleeding episodes in severe haemophilia A patients with inhibitors in Spain. A decision analytic model was designed to evaluate the costs and clinical outcomes of using rFVIIa or pd-aPCC to treat mild-to-moderate joint bleeds in children (≤14 years old) and adults with inhibitors. Data were obtained from a published meta-analysis and a panel of haemophilia experts. The analysis was conducted from the perspective of the Spanish National Healthcare System. One-way sensitivity analyses were performed to assess the impact of model assumptions on study results. In the Treur meta-analysis, rFVIIa resulted in cumulative joint bleed resolution of 88% and 95% after 24 and 36 h, respectively, compared with 62% and 76%, respectively, with pd-aPCC (Treur et al. Haemophilia 2009; 15: 420-36). Here, the mean cost per bleed was estimated at €8473 and €15 579 in children and adults treated with rFVIIa, vs. €8627 and €15 677 in children and adults treated with pd-aPCC. rFVIIa treatment was found to be the dominating option (cheaper and more effective). The one-way sensitivity analysis also confirmed that rFVIIa was less costly than pd-aPCC. The model suggests that rFVIIa is a cost-effective option compared with pd-aPCC for the treatment of mild-to-moderate bleeding episodes in a Spanish setting. © 2013 John Wiley & Sons Ltd.

Parents' information needs and influential factors when making decisions about TNF-α inhibitors.

PubMed

Lipstein, Ellen A; Lovell, Daniel J; Denson, Lee A; Kim, Sandra C; Spencer, Charles; Britto, Maria T

2016-09-15

Parents struggle when making treatment decisions for children with arthritis or other chronic conditions. Understanding their decision-making process is an essential step towards improving the decision-making experience. The objective of this study was to describe parents' information needs and the influences on their decision making about treatment with TNF-α inhibitors. Survey domains were developed based on qualitative data and cognitive interviewing. We mailed the survey to parents of children with juvenile idiopathic arthritis or inflammatory bowel disease who had initiated treatment with TNF-α inhibitors in the prior 2 years. Data were analyzed using descriptive and non-parametric statistics. Survey response rate was 54.9 %. Each item had <2 % missing responses. Parents used an array of information sources when deciding about treatment with TNF-α inhibitors. Resources other than their child's specialist were most often used to increase confidence in parents' decisions or because they wanted to know more about other people's experiences being treated with TNF-α inhibitors, rather than due to a lack of understanding. All but two (cost and route of administration) of the influential decision factors were very or extremely important to the majority of participants with factors related to long-term side effects, treatment efficacy, and disease impact being most important. This study describes parents' information needs and influential factors in treatment decision making. Results suggest that future work should be aimed at helping families weigh risks and benefits, such as through decision support interventions, as well as developing opportunities to include people beyond the family and physician in the decision-making process.

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

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

PubMed

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

2013-09-26

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.

Structure-activity relationships of hydroxamate-based histone deacetylase-8 inhibitors: reality behind anticancer drug discovery.

PubMed

Amin, Sk Abdul; Adhikari, Nilanjan; Jha, Tarun

2017-12-01

The pan-histone deacetylase (HDAC) inhibitors comprise a fish-like structural orientation where hydrophobic aryl- and zinc-binding groups act as head and tail, respectively of a fish. The linker moiety correlates the body of the fish linking head and tail groups. Despite these pan-HDAC inhibitors, selective HDAC-8 inhibitors are still in demand as a safe remedy. HDAC-8 is involved in invasion and metastasis in cancer. This review deals with the rationale behind HDAC-8 inhibitory activity and selectivity along with detailed structure-activity relationships of diverse hydroxamate-based HDAC-8 inhibitors. HDAC-8 inhibitory potency may be increased by modifying the fish-like pharmacophoric features of such type of pan-HDAC inhibitors. This review may provide a preliminary basis to design and optimize new lead molecules with higher HDAC-8 inhibitory activity. This work may surely enlighten in providing useful information in the field of target-specific anticancer therapy.

Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

PubMed

Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

2016-08-01

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

In vitro multifaceted activities of a specific group of novel phosphatidylinositol 3-kinase inhibitors on hotspot mutant PIK3CA.

PubMed

Kong, Dexin; Yamori, Takao; Yamazaki, Kanami; Dan, Shingo

2014-12-01

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in cancer, novel PI3K inhibitors such as ZSTK474, GDC-0941, NVP-BEZ235 and BKM-120 have been developed for cancer therapy. A high frequency of hotspot mutations known as E542K, E545K and H1047R in the PIK3CA gene, which encodes the catalytic subunit of PI3Kα, has been found in various types of human cancers. The hotspot PIK3CA mutations also lead to resistance to therapeutics targeting epidermal growth factor receptor (EGFR), further suggesting that inhibition of hotspot mutant PIK3CA be required for a PI3K inhibitor as anticancer drug candidate. To investigate the activity of the novel PI3K inhibitors on the hotspot mutant PIK3CA, we determined the inhibition against the respective recombinant mutant PI3Kαs by biochemical assay. We further examined the activity at cellular background by determining the effect on phosphorylation of Akt (Ser473), and that on the growth of cancer cells. In addition, apoptosis and autophagy in cells with or without hotspot PIK3CA mutation induced by the four inhibitors were investigated. Our results indicated that each inhibitor exhibit comparable activity on the hotspot mutant PI3Kα to that on the wild type, which was further demonstrated by the cell-based assays. No clear correlation was shown between the PIK3CA genetic status and the sensitivity for apoptosis or autophagy induction. Interestingly, among the 4 PI3K inhibitors, BKM-120 is the weakest in PI3K inhibitory potency, but induces most potent apoptosis, suggesting that BKM-120 might have a unique mode of action. Our result shows that the PI3K inhibitors exhibit potent activity on both hotspot mutant and wild type PI3Kα, suggesting they might be used to treat patients with or without PIK3CA mutation when approved.

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

PubMed Central

Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

2011-01-01

Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

PubMed

Vickers, Chris J; Olsen, Christian A; Leman, Luke J; Ghadiri, M Reza

2012-06-14

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

Lead finding for acetyl cholinesterase inhibitors from natural origin: structure activity relationship and scope.

PubMed

Mukherjee, P K; Satheeshkumar, N; Venkatesh, P; Venkatesh, M

2011-03-01

Acetylcholinesterase (AChE) inhibitors are considered as promising therapeutic agents for the treatment of several neurological disorders such as Alzheimer's disease (AD), senile dementia, ataxia and myasthenia gravis. There are only few synthetic medicines with adverse effects, available for treatment of cognitive dysfunction and memory loss associated with these diseases. A variety of plants has been reported to possess AChE inhibitory activity and so may be relevant to the treatment of neurodegenerative disorders such as AD. Hence, developing potential AChE inhibitors from botanicals is the need of the day. This review will cover some of the promising acetylcholinesterase inhibitors isolated from plants with proven in vitro and in vivo activities with concern to their structure activity relationship.

NEW RENIN INHIBITORS - STABILITY AND ACTIVITY DETERMINATION. PART IV.

PubMed

Marszalek, Dorota; Goldnik, Anna; Winiecka, Iwona; Jaworsk, Pawel; Mazurek Aleksander P

2017-03-01

A series of new seven potential renin inhibitors containing pseudodipeptides were synthesized. Stability for all compounds (1-7) in homogenates of liver, kidney, lung and in serum, gastric, intestinal juice and in the presence of α-chymotrypsin was determined. Compound 1 was unstable in all determined mediums. Compounds 2, 4, 5 and 7 were unstable, compound 3 was stable, compound 6 was unstable only in α-chy-motrypsin solution. Inhibitory activity of the compounds was measured in vitro by HPLC determination of low-ering concentration of substrate (angiotensinogen) in the presence of renin and the potential renin inhibitor (compounds 1-7). Compounds 1, 2, 4, 5, 6 and 7 showed inhibitory activity (1.12 x 10⁻⁷, 0.96 x 10⁻⁶, 1.58 x10⁻⁷,1.68 x 10⁻⁶, 1.30 x 10⁻⁶, 0.96 x 10⁻⁷M, respectively).

The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheffield, William P., E-mail: sheffiel@mcmaster.ca; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario; Bhakta, Varsha

The naturally occurring M358R mutation of the plasma serpin α{sub 1}-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 assaysmore » 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{sup 2} M{sup −1}sec{sup −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. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.« less

Targeted Morphoproteomic Profiling of Ewing's Sarcoma Treated with Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors: Response/Resistance Signatures

PubMed Central

Subbiah, Vivek; Naing, Aung; Brown, Robert E.; Chen, Helen; Doyle, Laurence; LoRusso, Patricia; Benjamin, Robert; Anderson, Pete; Kurzrock, Razelle

2011-01-01

Background Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing's sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing's sarcoma to IGF1R inhibitor-based therapy. Methodology/Principal Findings This pilot study assessed two patients with advanced Ewing's sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well. Conclusion/Significance Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing's sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing's sarcoma merits further investigation as a guide to understanding response and resistance signatures. PMID

Dipeptidyl Peptidase-4 Inhibitor Anagliptin Prevents Intracranial Aneurysm Growth by Suppressing Macrophage Infiltration and Activation.

PubMed

Ikedo, Taichi; Minami, Manabu; Kataoka, Hiroharu; Hayashi, Kosuke; Nagata, Manabu; Fujikawa, Risako; Higuchi, Sei; Yasui, Mika; Aoki, Tomohiro; Fukuda, Miyuki; Yokode, Masayuki; Miyamoto, Susumu

2017-06-19

Chronic inflammation plays a key role in the pathogenesis of intracranial aneurysms (IAs). DPP-4 (dipeptidyl peptidase-4) inhibitors have anti-inflammatory effects, including suppressing macrophage infiltration, in various inflammatory models. We examined whether a DPP-4 inhibitor, anagliptin, could suppress the growth of IAs in a rodent aneurysm model. IAs were surgically induced in 7-week-old male Sprague Dawley rats, followed by oral administration of 300 mg/kg anagliptin. We measured the morphologic parameters of aneurysms over time and their local inflammatory responses. To investigate the molecular mechanisms, we used lipopolysaccharide-treated RAW264.7 macrophages. In the anagliptin-treated group, aneurysms were significantly smaller 2 to 4 weeks after IA induction. Anagliptin inhibited the accumulation of macrophages in IAs, reduced the expression of MCP-1 (monocyte chemotactic protein 1), and suppressed the phosphorylation of p65. In lipopolysaccharide-stimulated RAW264.7 cells, anagliptin treatment significantly reduced the production of tumor necrosis factor α, MCP-1, and IL-6 (interleukin 6) independent of GLP-1 (glucagon-like peptide 1), the key mediator in the antidiabetic effects of DPP-4 inhibitors. Notably, anagliptin activated ERK5 (extracellular signal-regulated kinase 5), which mediates the anti-inflammatory effects of statins, in RAW264.7 macrophages. Preadministration with an ERK5 inhibitor blocked the inhibitory effect of anagliptin on MCP-1 and IL-6 expression. Accordingly, the ERK5 inhibitor also counteracted the suppression of p65 phosphorylation in vitro. A DPP-4 inhibitor, anagliptin, prevents the growth of IAs via its anti-inflammatory effects on macrophages. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

PubMed Central

Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2010-01-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

PubMed Central

2016-01-01

Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng Jianbei; Hua Kunjie; Caveney, Erica J.

2006-01-20

Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in themore » cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.« less

Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas.

PubMed

Roskoski, Robert

2017-06-01

One Von Hippel-Lindau (VHL) tumor suppressor gene is lost in most renal cell carcinomas while the nondeleted allele exhibits hypermethylation-induced inactivation or inactivating somatic mutations. As a result of these genetic modifications, there is an increased production of VEGF-A and pro-angiogenic growth factors in this disorder. The important role of angiogenesis in the pathogenesis of renal cell carcinomas and other tumors has focused the attention of investigators on the biology of VEGFs and VEGFR1-3 and to the development of inhibitors of the intricate and multifaceted angiogenic pathways. VEGFR1-3 contain an extracellular segment with seven immunoglobulin-like domains, a transmembrane segment, a juxtamembrane segment, a protein kinase domain with an insert of about 70 amino acid residues, and a C-terminal tail. VEGF-A stimulates the activation of preformed VEGFR2 dimers by the auto-phosphorylation of activation segment tyrosines followed by the phosphorylation of additional protein-tyrosines that recruit phosphotyrosine binding proteins thereby leading to signalling by the ERK1/2, AKT, Src, and p38 MAP kinase pathways. VEGFR1 modulates the activity of VEGFR2, which is the chief pathway in vasculogenesis and angiogenesis. VEGFR3 and its ligands (VEGF-C and VEGF-D) are involved primarily in lymphangiogenesis. Small molecule VEGFR1/2/3 inhibitors including axitinib, cabozantinib, lenvatinib, sorafenib, sunitinib, and pazopanib are approved by the FDA for the treatment of renal cell carcinomas. Most of these agents are type II inhibitors of VEGFR2 and inhibit the so-called DFG-Asp out inactive enzyme conformation. These drugs are steady-state competitive inhibitors with respect to ATP and like ATP they form hydrogen bonds with the hinge residues that connect the small and large protein kinase lobes. Bevacizumab, a monoclonal antibody that binds to VEGF-A, is also approved for the treatment of renal cell carcinomas. Resistance to these agents invariably occurs

Click Chemistry-based Discovery of [3-Hydroxy-5-(1H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of Anemia.

PubMed

Wu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qi-Dong; Zhang, Xiaojin

2018-06-01

As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assay. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.

Sarcoidosis as an adverse effect of tumor necrosis factor inhibitors.

PubMed

Cathcart, Shelley; Sami, Naveed; Elewski, Boni

2012-05-01

Tumor necrosis factor inhibitors are valuable tools for dermatologists. As their use increases, rare adverse events are more likely to be encountered. We describe one patient who developed sarcoidosis while being treated for psoriasis with etanercept. We sought to review to previously reported cases and further characterize the nature of this reaction. A literature search was performed with the key words "sarcoidosis, sarcoid, etanercept, infliximab, adalimumab, granulomatous, and drug reaction." All relevant cases in the English language were included and evaluated for demographic data, duration of therapy prior to developing sarcoid, duration of sarcoid signs/symptoms, treatments used and time to resolution after discontinuation of the drug. Including the present case, there are 34 cases of sarcoidosis developing during anti-tumor necrosis factor therapy. All previously reported cases were patients with a primarily rheumatologic diagnosis. In all but one case, discontinuation of the drug resulted in complete resolution of symptoms. The lung and surrounding lymph nodes were the areas most commonly affected. The average amount of time between initiation of therapy and onset of symptoms was 22 months. The average time to resolution of symptoms after discontinuation of the drug was 5.2 months. This is a retrospective case review. These data indicated that sarcoid is a possible adverse effect of tumor necrosis factor inhibitor therapy that should be noted by dermatologists using these drugs. While it has been reported in the rheumatology literature, it may be under-recognized by dermatologists.

Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396.

PubMed

Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K; Westwood, Nicholas J; Adamson, Catherine S

2016-09-15

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they

Safety of 4-factor prothrombin complex concentrate (4F-PCC) for emergent reversal of factor Xa inhibitors.

PubMed

Tao, Jing; Bukanova, Elena N; Akhtar, Shamsuddin

2018-01-01

Although factor Xa inhibitors have become a popular choice for chronic oral anticoagulation, effective drug reversal remains difficult due to a lack of specific antidote. Currently, 4-factor prothrombin complex concentrate (4F-PCC) is considered the treatment of choice for factor Xa inhibitor-related major bleeding. However, safety of 4F-PCC and its risk of thrombosis when used for this off-label purpose remain unclear. The purpose of this retrospective study is to determine the rate of thromboembolism when 4F-PCC is used for the emergent reversal of factor Xa inhibitors. We conducted a single-center retrospective review of medical records between 2013 and 2017. Patients were included if they received 4F-PCC to reverse rivaroxaban, apixaban, or edoxaban for emergent invasive procedures or during episodes of major bleeding defined as bleeding with hemodynamic instability, fall in hemoglobin of 2 g/dL, or bleeding requiring blood transfusion. Thrombotic events including myocardial infarction, pulmonary embolism, deep vein thrombosis, cerebral vascular accident, and arterial thrombosis of the limb or mesentery were recorded if they occurred within 14 days of 4F-PCC administration. Data was analyzed using point and interval estimation to approximate the rate and confidence interval of thromboembolic events. Forty-three patients were identified in our review. Doses of 4F-PCC were determined by the treating physician and mainly ranged from 25 to 50 IU/kg. Twenty-two patients (51.2%) received both sequential compression devices (SCDs) and subcutaneous heparin for DVT prophylaxis. Twenty-one patients (48.8%) were placed on SCDs only. Three patients received concomitant FFP. Thrombotic events within 14 days of 4F-PCC administration occurred in 1 out of 43 patients (2.1%, 95% CI [0.1-12.3]). This thrombotic event was an upper extremity DVT which occurred 1 day after the patient received 1325 IU (25 IU/kg) of 4F-PCC to reverse rivaroxaban for traumatic intracranial

Target Residence Time-Guided Optimization on TTK Kinase Results in Inhibitors with Potent Anti-Proliferative Activity.

PubMed

Uitdehaag, Joost C M; de Man, Jos; Willemsen-Seegers, Nicole; Prinsen, Martine B W; Libouban, Marion A A; Sterrenburg, Jan Gerard; de Wit, Joeri J P; de Vetter, Judith R F; de Roos, Jeroen A D M; Buijsman, Rogier C; Zaman, Guido J R

2017-07-07

The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nontraumatic spinal subdural hematoma complicating direct factor Xa inhibitor treatment (rivaroxaban): a challenging management.

PubMed

Dargazanli, Cyril; Lonjon, Nicolas; Gras-Combe, Guillaume

2016-05-01

We report on a 72-year-old male patient who developed a nontraumatic spinal subdural hematoma (SSDH) during rivaroxaban therapy, a relatively new orally administered direct factor Xa inhibitor. The patient sustained a sudden onset of interscapular pain, followed by gait impairment and paraplegia. Magnetic resonance imaging (MRI) of the spine demonstrated SSDH from T6 to T8. Laboratory tests revealed a high rivaroxaban level, associated with a major hemorrhagic risk. Surgery was, therefore, performed the following morning, after normalization of coagulation parameters. Determining the time of safe surgery remains challenging when hemorrhagic complications happen with direct factor Xa inhibitor, especially when neurological prognosis is engaged. Spinal subdural hematoma has not previously been reported following rivaroxaban therapy.

Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity.

PubMed

El-Asrar, Mohamed Abo; Hamed, Ahmed El-Saeed; Darwish, Yasser Wagih; Ismail, Eman Abdel Rahman; Ismail, Noha Ali

2016-01-01

A rapidly growing evidence showed that regulatory T cells (Tregs) play a crucial role in tolerance to coagulation factors and may be involved in the pathogenesis of inhibitor formation in patients with hemophilia. We determined the percentage of Tregs (CD4CD25CD127) in 45 children with hemophilia A compared with 45 healthy controls, and assessed their relation to the clinical characteristics of patients and factor VIII (FVIII) inhibitors. Patients were studied stressing on frequency of bleeding attacks, joint pain, history of viral hepatitis, and the received therapy (FVIII precipitate/cryotherapy). FVIII activity and FVIII inhibitors were assessed with flow cytometric analysis of CD4CD25CD127 Tregs. According to residual FVIII activity levels, 30 patients (66.7%) had mild/moderate hemophilia A, whereas 15 (33.3%) patients had severe hemophilia A. The frequency of Tregs was significantly lower among all patients with hemophilia A compared with controls (2.59 ± 1.1 versus 3.73 ± 1.12%; P = 0.002). Tregs were significantly decreased among patients with FVIII inhibitors compared with the inhibitor-negative group (P < 0.001). Patients with hematuria or severe hemophilia A had lower Tregs levels than those without (P = 0.34 and P = 0.011, respectively). A significant positive correlation was found between the percentage of Tregs and FVIII among hemophilia A patients. ROC curve analysis revealed that the cut-off value of Tregs at 1.91% could differentiate patients with and without FVIII inhibitors, with a sensitivity of 100% and a specificity of 91.3%. We suggest that alteration in the frequency of Tregs in young patients with hemophilia A may contribute to inhibitor formation and disease severity.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

PubMed Central

Baker, Emma K; Taylor, Scott; Gupte, Ankita; Sharp, Phillip P; Walia, Mannu; Walsh, Nicole C; Zannettino, Andrew CW; Chalk, Alistair M; Burns, Christopher J; Walkley, Carl R

2015-01-01

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS. PMID:25944566

Factor Xa inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in patients with atrial fibrillation.

PubMed

Bruins Slot, Karsten M H; Berge, Eivind

2013-08-08

Anticoagulant treatment with vitamin K antagonists (VKAs) is aimed at preventing thromboembolic complications and has been the therapy of choice for most people with non-valvular atrial fibrillation (AF) for many decades. A new class of anticoagulants, the factor Xa inhibitors, appear to have several pharmacological and practical advantages over VKAs. To assess the effectiveness and safety of treatment with factor Xa inhibitors versus VKAs for the prevention of cerebral or systemic embolic events in people with AF. We searched the trials registers of the Cochrane Stroke Group and the Cochrane Heart Group (June 2012), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, Issue 10), MEDLINE (1950 to April 2013) and EMBASE (1980 to April 2013). In an effort to identify further published, unpublished and ongoing trials we searched trials registers and Google Scholar (July 2012). We also screened reference lists and contacted pharmaceutical companies, authors and sponsors of relevant published trials. Randomised controlled trials that directly compared the effects of long-term treatment (more than four weeks) with factor Xa inhibitors and VKAs for the prevention of cerebral and systemic embolism in patients with AF. We included patients with and without a previous stroke or TIA. The primary efficacy outcome was the composite endpoint of all strokes and other systemic embolic events. Two authors independently assessed trial quality and the risk of bias, and extracted data. We calculated a weighted estimate of the typical treatment effect across trials using the odds ratio (OR) with 95% confidence interval (CI) by means of a fixed-effect model. However, in the case of moderate or high heterogeneity of treatment effects, we used a random-effects model to compare the overall treatment effects and performed a pre-specified sensitivity analysis excluding any fully open-label studies. We included data from 42,084 participants randomised into

Tissue factor pathway inhibitor, activated protein C resistance, and risk of ischemic stroke due to postmenopausal hormone therapy

PubMed Central

Rossouw, Jacques E; Johnson, Karen C; Pettinger, Mary; Cushman, Mary; Sandset, Per Morten; Kuller, Lewis; Rosendaal, Frits; Rosing, Jan; Wasserthal-Smoller, Sylvia; Martin, Lisa W; Manson, JoAnn E; Lakshminarayan, Kamakshi; Merino, Jose G; Lynch, John

2012-01-01

Background and Purpose To test whether changes in plasma tissue factor pathway inhibitor (TFPI) levels or activated protein C resistance (normalized APC resistance ratio, nAPCsr) modify the increased risk of ischemic stroke due to postmenopausal hormone therapy (PHT). Methods Nested case-control study of 455 cases of ischemic stroke and 565 matched controls in the Women’s Health Initiative trials of PHT. Results Baseline free TFPI was associated with ischemic stroke risk, OR (95% CI) per SD increase = 1.17 (1.01, 1.37, p=0.039, but baseline nAPCsr was not, OR per SD increase = 0.89 (0.75, 1.05), p=0.15. Baseline TFPI levels and nAPCsr did not modify the effect of PHT on ischemic stroke. Treatment-induced mean changes of -28% in free TFPI and +65% in nAPCsr did not change the risk of ischemic stroke (interaction p = 0.452 and 0.971 respectively). In subgroup analyses baseline nAPCsr was inversely associated with lacunar strokes, OR per SD increase = 0.74 (0.57, 0.96), p=0.025, and baseline free TFPI interacted with treatment to increase large vessel atherosclerotic strokes, p=0.008. Conclusions Pro-coagulant changes in TFPI or nAPCsr do not modify the increased ischemic stroke risk due to PHT. PMID:22363056

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

PubMed Central

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Jie; Zeng, Li-Fan; Shen, Weihua

Highlights: •SHP2 is required for EGFR inhibitor resistant NSCLC H1975 cell proliferation. •SHP2 inhibitor blocks EGF-stimulated ERK1/2 activation and proliferation. •SHP2 inhibitor exhibits marked anti-tumor activity in H1975 xenograft mice. •SHP2 inhibitor synergizes with PI3K inhibitor in suppressing cell growth. •Targeting SHP2 represents a novel strategy for EGFR inhibitor resistant NSCLCs. -- Abstract: Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFRmore » (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.« less

Factor Xa inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in patients with atrial fibrillation.

PubMed

Bruins Slot, Karsten Mh; Berge, Eivind

2018-03-06

Factor Xa inhibitors and vitamin K antagonists (VKAs) are now recommended in treatment guidelines for preventing stroke and systemic embolic events in people with atrial fibrillation (AF). This is an update of a Cochrane review previously published in 2013. To assess the effectiveness and safety of treatment with factor Xa inhibitors versus VKAs for preventing cerebral or systemic embolic events in people with AF. We searched the trials registers of the Cochrane Stroke Group and the Cochrane Heart Group (September 2016), the Cochrane Central Register of Controlled Trials (CENTRAL) (August 2017), MEDLINE (1950 to April 2017), and Embase (1980 to April 2017). We also contacted pharmaceutical companies, authors and sponsors of relevant published trials. We used outcome data from marketing authorisation applications of apixaban, edoxaban and rivaroxaban that were submitted to regulatory authorities in Europe and the USA. We included randomised controlled trials (RCTs) that directly compared the effects of long-term treatment (lasting more than four weeks) with factor Xa inhibitors versus VKAs for preventing cerebral and systemic embolism in people with AF. The primary efficacy outcome was the composite endpoint of all strokes and systemic embolic events. Two review authors independently extracted data, and assessed the quality of the trials and the risk of bias. We calculated a weighted estimate of the typical treatment effect across trials using the odds ratio (OR) with 95% confidence interval (CI) by means of a fixed-effect model. In case of moderate or high heterogeneity of treatment effects, we used a random-effects model to compare the overall treatment effects. We also performed a pre-specified sensitivity analysis excluding any open-label studies. We included data from 67,688 participants randomised into 13 RCTs. The included trials directly compared dose-adjusted warfarin with either apixaban, betrixaban, darexaban, edoxaban, idraparinux, idrabiotaparinux, or

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

PubMed Central

Cencic, Regina; Pelletier, Jerry

2016-01-01

ABSTRACT 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

StInvInh2 as an inhibitor of StvacINV1 regulates the cold-induced sweetening of potato tubers by specifically capping vacuolar invertase activity.

PubMed

Liu, Xun; Lin, Yuan; Liu, Jun; Song, Botao; Ou, Yongbin; Zhang, Huiling; Li, Meng; Xie, Conghua

2013-06-01

Reducing sugar (RS) accumulation in cold-stored potato tubers, known as cold-induced sweetening (CIS), is a crucial factor causing unacceptable colour changes and acrylamide formation of fried products. The activity of vacuolar invertase (StvacINV1) is proved important for the CIS process, and invertase inhibitors are speculated to play roles in the post-translational regulation of StvacINV1 activity. In our previous research, two putative inhibitors (StInvInh2A and StInvInh2B) of StvacINV1 were implied to be involved in potato CIS. Here, we further reported that StInvInh2A and StInvInh2B had similar function that specifically inhibited StvacINV1 activity in potatoes. The genetic transformation of these inhibitor genes in potatoes by overexpression in CIS-sensitive and RNAi-silenced in CIS-resistant genotypes showed that StvacINV1 activity was strongly regulated by alteration of the transcripts of the inhibitors without impacting on the expression of StvacINV1. A negative power relationship was found between the transcripts of the inhibitors and StvacINV1 activity, suggesting 1) a transcriptional determination of the inhibitory capacity of StInvInh2A and StInvInh2B and 2) a significant inhibitory role of these inhibitors in post-translational modulation of StvacINV1. The results also demonstrated that depression of StvacINV1 activity through overexpression of StInvInh2A and StInvInh2B weakened accumulation of RS and acrylamide in cold-stored tubers and consequently improved the chip quality. The present research strongly suggest that both StInvInh2A and StInvInh2B function as inhibitors of StvacINV1 and play similar roles in regulating potato CIS by capping StvacINV1 activity. These inhibitors could be novel genetic resources applicable for improving quality of potato processing products. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Kinome and Transcriptome Profiling Reveal Broad and Distinct Activities of Erlotinib, Sunitinib, and Sorafenib in the Mouse Heart and Suggest Cardiotoxicity From Combined Signal Transducer and Activator of Transcription and Epidermal Growth Factor Receptor Inhibition.

PubMed

Stuhlmiller, Timothy J; Zawistowski, Jon S; Chen, Xin; Sciaky, Noah; Angus, Steven P; Hicks, Sean T; Parry, Traci L; Huang, Wei; Beak, Ju Youn; Willis, Monte S; Johnson, Gary L; Jensen, Brian C

2017-10-19

Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

PubMed

Wiggers, Helton J; Rocha, Josmar R; Fernandes, William B; Sesti-Costa, Renata; Carneiro, Zumira A; Cheleski, Juliana; da Silva, Albérico B F; Juliano, Luiz; Cezari, Maria H S; Silva, João S; McKerrow, James H; Montanari, Carlos A

2013-01-01

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound

Inhibitor of Nicotinamide Phosphoribosyltransferase Sensitizes Glioblastoma Cells to Temozolomide via Activating ROS/JNK Signaling Pathway.

PubMed

Feng, Jun; Yan, Peng-Fei; Zhao, Hong-Yang; Zhang, Fang-Cheng; Zhao, Wo-Hua; Feng, Min

2016-01-01

Overcoming temozolomide (TMZ) resistance is a great challenge in glioblastoma (GBM) treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide and has a crucial role in cancer cell metabolism. In this study, we investigated whether FK866 and CHS828, two specific NAMPT inhibitors, could sensitize GBM cells to TMZ. Low doses of FK866 and CHS828 (5 nM and 10 nM, resp.) alone did not significantly decrease cell viability in U251-MG and T98 GBM cells. However, they significantly increased the antitumor action of TMZ in these cells. In U251-MG cells, administration of NAMPT inhibitors increased the TMZ (100  μ M)-induced apoptosis and LDH release from GBM cells. NAMPT inhibitors remarkably enhanced the activities of caspase-1, caspase-3, and caspase-9. Moreover, NAMPT inhibitors increased reactive oxygen species (ROS) production and superoxide anion level but reduced the SOD activity and total antioxidative capacity in GBM cells. Treatment of NAMPT inhibitors increased phosphorylation of c-Jun and JNK. Administration of JNK inhibitor SP600125 or ROS scavenger tocopherol with TMZ and NAMPT inhibitors substantially attenuated the sensitization of NAMPT inhibitor on TMZ antitumor action. Our data indicate a potential value of NAMPT inhibitors in combined use with TMZ for GBM treatment.

Aceroside VIII is a new natural selective HDAC6 inhibitor that synergistically enhances the anticancer activity of HDAC inhibitor in HT29 cells.

PubMed

Ryu, Hyun-Wook; Lee, Dong-Hun; Shin, Dong-Hee; Kim, Seung Hyun; Kwon, So Hee

2015-02-01

The identification of new isoform-specific histone deacetylase inhibitors is important for revealing the biological functions of individual histone deacetylase and for determining their potential use as therapeutic agents. Among the 11 zinc-dependent histone deacetylases that have been identified in humans, histone deacetylase 6 is a structurally and functionally unique enzyme. Here, we tested the inhibitory activity of diarylheptanoids isolated from Betula platyphylla against histone deacetylase 6. Aceroside VIII selectively inhibited histone deacetylase 6 catalytic activity and the combined treatment of aceroside VIII or (-)-centrolobol with A452, another selective histone deacetylase 6 inhibitor, led to a synergistic increase in levels of acetylated α-tubulin. Aceroside VIII, paltyphyllone, and (-)-centrolobol synergistically enhanced the induction of apoptosis and growth inhibition by A452. Consistent with these results, A452 in combination with aceroside VIII, paltyphyllone, or (-)-centrolobol was more potent than either drug alone for the induction of apoptosis. Together, these findings indicate that aceroside VIII is a specific histone deacetylase 6 inhibitor and points to a mechanism by which natural histone deacetylase 6-selective inhibitors may enhance the efficacy of other histone deacetylase 6 inhibitors in colon cancer cells. Georg Thieme Verlag KG Stuttgart · New York.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiao,J.; Chang, C.; Cheney, D.

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.

Rho-associated kinase inhibitors: a novel glaucoma therapy.

PubMed

Inoue, Toshihiro; Tanihara, Hidenobu

2013-11-01

The rho-associated kinase (ROCK) signaling pathway is activated via secreted bioactive molecules or via integrin activation after extracellular matrix binding. These lead to polymerization of actin stress fibers and formation of focal adhesions. Accumulating evidence suggests that actin cytoskeleton-modulating signals are involved in aqueous outflow regulation. Aqueous humor contains various biologically active factors, some of which are elevated in glaucomatous eyes. These factors affect aqueous outflow, in part, through ROCK signaling modulation. Various drugs acting on the cytoskeleton have also been shown to increase aqueous outflow by acting directly on outflow tissue. In vivo animal studies have shown that the trabecular meshwork (TM) actin cytoskeleton in glaucomatous eyes is more disorganized and more randomly oriented than in non-glaucomatous control eyes. In a previous study, we introduced ROCK inhibitors as a potential glaucoma therapy by showing that a selective ROCK inhibitor significantly lowered rabbit IOP. Rho-associated kinase inhibitors directly affect the TM and Schlemm's canal (SC), differing from the target sight of other glaucoma drugs. The TM is affected earlier and more strongly than ciliary muscle cells by ROCK inhibitors, largely because of pharmacological affinity differences stemming from regulatory mechanisms. Additionally, ROCK inhibitors disrupt tight junctions, result in F-actin depolymerization, and modulate intracellular calcium level, effectively increasing SC-cell monolayer permeability. Perfusion of an enucleated eye with a ROCK inhibitor resulted in wider empty spaces in the juxtacanalicular (JCT) area and more giant vacuoles in the endothelial cells of SC, while the endothelial lining of SC was intact. Interestingly, ROCK inhibitors also increase retinal blood flow by relaxing vascular smooth muscle cells, directly protecting neurons against various stresses, while promoting wound healing. These additional effects may help

Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs inhibitors.

PubMed

Liu, Yang; Peng, Xia; Guan, Xiaocong; Lu, Dong; Xi, Yong; Jin, Shiyu; Chen, Hui; Zeng, Limin; Ai, Jing; Geng, Meiyu; Hu, Youhong

2017-01-27

FGF receptors (FGFRs) are tyrosine kinases that are overexpressed in diverse tumors by genetic alterations such as gene amplifications, somatic mutations and translocations. Owing to this characteristic, FGFRs are attractive targets for cancer treatment. It has been demonstrated that most multi-targeted, ATP competitive tyrosine kinase inhibitors are active against FGFRs as well as other kinases. The design of new and more selective inhibitors of FGFRs, which might be reduced off-target and side effects, is a difficult yet significant challenge. The results of the current investigation, show that novel Ponatinib analogues are highly active as FGFR inhibitors and that they possess reduced kinase insert domain receptor (KDR) activities. Observations made in a structure and activity relationship (SAR) investigation led to the development of a promising, orally available lead compound 4, which displays a 50-100 fold in vitro selectivity for inhibition of FGFR1-3 over KDR. In addition, biological evaluation of compound 4 showed that it displays significant antitumor activities in FGFR1-amplificated H1581 and FGFR2-amplificated SNU-16 xenograft models. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effects of tumor necrosis factor inhibitor on serum level of HLA-B27 and PDCD-1 in patients with ankylosing spondylitis.

PubMed

Chen, Xiaogang; Zhou, Xiaoqing; Li, Xia; Tang, Jinshan; Hu, Xiaowu; Wang, Junsheng; Xu, Cheng

2014-11-01

The aim of this study was to evaluate the effect of tumor necrosis factor-alpha (TNF-α) inhibitor-infliximab on ankylosing spondylitis (AS) patients and detect the serum level of HLA-B27 and PDCD-1 before and after TNF inhibitor treatment. 138 patients at active stage of AS were treated with infliximab; serum was collected before and after TNF-α inhibitor treatment for analysis. Reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and enzyme-linked immuno sorbent assay were applied to detect the levels of HLA-B27 and PDCD-1 at different time points, which were used for statistical analysis with clinical data including two AS indicators (erythrocyte sedimentation rate--ESR and C-reactive protein--CRP). After the treatment for 6 weeks, RT-PCR showed that the gene expressions of HLA-B27 and PDCD-1 were significantly downregulated compared with baseline before infliximab treatment (P < 0.05); flow cytometry showed that the HLA-B27 and PDCD-1 double-labeled cells were significantly downregulated (P < 0.05). After 2, 6, or 10 weeks of infliximab treatment, the levels of ESR, CRP, serum HLA-B27, and PDCD-1 of the AS patients were all significantly lower than the baseline levels (P < 0.05), and the serum HLA-B27 and PDCD-1 levels were all significantly correlated with ESR (P < 0.05). Infliximab, an anti-TNF-α inhibitor, decreases significantly not only ESR and CRP, but also the serum levels of HLA-B27 and PDCD-1 in patients with AS. HLA-B27 and PDCD-1 are involved in the pathogenesis, and disease activities of AS. HLA-B27 and PDCD-1 are potentially the useful markers of AS activity and useful parameters to evaluate the effectiveness of anti-TNF-α inhibitor in treating AS.

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

NASA Astrophysics Data System (ADS)

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.

Activated MET acts as a salvage signal after treatment with alectinib, a selective ALK inhibitor, in ALK-positive non-small cell lung cancer.

PubMed

Kogita, Akihiro; Togashi, Yosuke; Hayashi, Hidetoshi; Banno, Eri; Terashima, Masato; De Velasco, Marco A; Sakai, Kazuko; Fujita, Yoshihiko; Tomida, Shuta; Takeyama, Yoshifumi; Okuno, Kiyotaka; Nakagawa, Kazuhiko; Nishio, Kazuto

2015-03-01

Non-small cell lung cancer (NSCLC) carrying echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements is hypersensitive to ALK inhibitors, including crizotinib and alectinib. Crizotinib was initially designed as a MET inhibitor, whereas alectinib is a selective ALK inhibitor. The MET signal, which is inhibited by crizotinib but not by alectinib, is dysregulated in many human cancers. However, the role of the MET signal in ALK-positive NSCLC remains unclear. In this study, we found that hepatocyte growth factor (HGF), ligand of MET, mediated the resistance to alectinib, but not to crizotinib, via the MET signal in ALK-positive NSCLC cell lines (H3122 and H2228 cell lines). In addition, alectinib activated the MET signal even in the absence of HGF and the inhibition of the MET signal enhanced the efficacy of alectinib. These findings suggest that activated MET acts as a salvage signal in ALK-positive NSCLC. This novel role of the MET signal in ALK-positive NSCLC may pave the way for further clinical trials examining MET inhibitors.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Role of activator protein-1 on the effect of arginine-glycine-aspartic acid containing peptides on transforming growth factor-beta1 promoter activity.

PubMed

Ruiz-Torres, M P; Perez-Rivero, G; Diez-Marques, M L; Griera, M; Ortega, R; Rodriguez-Puyol, M; Rodríguez-Puyol, D

2007-01-01

While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.

Effect of ripening inhibitor type on formation, stability, and antimicrobial activity of thyme oil nanoemulsion.

PubMed

Ryu, Victor; McClements, David J; Corradini, Maria G; McLandsborough, Lynne

2018-04-15

The objective of this research was to study the impact of ripening inhibitor level and type on the formation, stability, and activity of antimicrobial thyme oil nanoemulsions formed by spontaneous emulsification. Oil-in-water antimicrobial nanoemulsions (10 wt%) were formed by titrating a mixture of essential oil, ripening inhibitor, and surfactant (Tween 80) into 5 mM sodium citrate buffer (pH 3.5). Stable nanoemulsions containing small droplets (d < 70 nm) were formed. The antimicrobial activity of the nanoemulsions decreased with increasing ripening inhibitor concentration which was attributed to a reduction in the amount of hydrophobic antimicrobial constituents transferred to the separated hydrophobic domain, mimicking bacterial cell membranes, by using dialysis and chromatography. The antimicrobial activity of the nanoemulsions also depended on the nature of the ripening inhibitor used: palm ≈ corn > canola > coconut which also depended on their ability to transfer hydrophobic antimicrobial constituents to the separated hydrophobic domain. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance.

PubMed

Hu, Hsien-Ming; Zhao, Xin; Kaushik, Swati; Robillard, Lilliane; Barthelet, Antoine; Lin, Kevin K; Shah, Khyati N; Simmons, Andy D; Raponi, Mitch; Harding, Thomas C; Bandyopadhyay, Sourav

2018-04-17

Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

PubMed

Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

2012-12-01

Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

Design, Synthesis, and Herbicidal Activity of Pyrimidine-Biphenyl Hybrids as Novel Acetohydroxyacid Synthase Inhibitors.

PubMed

Li, Ke-Jian; Qu, Ren-Yu; Liu, Yu-Chao; Yang, Jing-Fang; Devendar, Ponnam; Chen, Qiong; Niu, Cong-Wei; Xi, Zhen; Yang, Guang-Fu

2018-04-18

The issue of weed resistance to acetohydroxyacid synthase (EC 2.2.1.6, AHAS) inhibitors has become one of the largest obstacles for the application of this class of herbicides. In a continuing effort to discover novel AHAS inhibitors to overcome weed resistance, a series of pyrimidine-biphenyl hybrids (4aa-bb and 5aa-ah) were designed and synthesized via a scaffold hopping strategy. Among these derivatives, compounds 4aa ( K i = 0.09 μM) and 4bb ( K i = 0.02 μM) displayed higher inhibitory activities against Arabidopsis thaliana AHAS than those of the controls bispyribac ( K i = 0.54 μM) and flumetsulam ( K i = 0.38 μM). Remarkably, compounds 4aa, 4bb, 5ah, and 5ag exhibited excellent postemergence herbicidal activity and a broad spectrum of weed control at application rates of 37.5-150 g of active ingredient (ai)/ha. Furthermore, 4aa and 4bb showed higher herbicidal activity against AHAS inhibitor-resistant Descurainia sophia, Ammannia arenaria, and the corresponding sensitive weeds than that of bispyribac at 0.94-0.235 g ai/ha. Therefore, the pyrimidine-biphenyl motif and lead compounds 4aa and 4bb have great potential for the discovery of novel AHAS inhibitors to combat AHAS-inhibiting herbicide-resistant weeds.

HER receptor signaling confers resistance to the insulin-like growth factor 1 receptor inhibitor, BMS-536924

PubMed Central

Haluska, Paul; Carboni, Joan M.; Eyck, Cynthia Ten; Attar, Ricardo M.; Hou, Xiaonan; Yu, Chunrong; Sagar, Malvika; Wong, Tai W.; Gottardis, Marco M.; Erlichman, Charles

2008-01-01

We have previously reported the activity of the IGF-1R/InsR inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER2. In addition, treatment with the panHER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF1-R, suggesting a reciprocal crosstalk mechanism. In a panel of five ovarian cancer cell lines simultaneous treatment with the IGF-1R/InsR inhibitor, BMS-536924 and BMS-599626 resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and ERK activation and increased biochemical and nuclear morphological changes consistent with apoptosis as compared to either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting inhibition of IGF-1R/InsR results in adaptive upregulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER1 or HER2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-1R targeted therapy. In the presence of activating ligands EGF or heregulin, respectively, MCF-7 cells expressing HER1 or HER2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-1 and HER family of receptors may be an effective strategy for clinical investigations of IGF-1R inhibitors in breast and ovarian cancer and that targeting HER1 and HER2 may overcome clinical resistance to IGF-1R inhibitors. PMID:18765823

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moyer, Benjamin J.

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase genemore » reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes. - Highlights: • Indoleamine-2,3-dioxygenase 1 (IDO1) inhibitors are in cancer clinical trials. • Some IDO1 inhibitors also potently activate AHR signaling. • The dual role of the IDO1 inhibitors may explain some past paradoxical findings. • AHR induction studies must be included in assessing clinical suitability.« less

Fast-forwarding hit to lead: aurora and epidermal growth factor receptor kinase inhibitor lead identification.

PubMed

Coumar, Mohane Selvaraj; Chu, Chang-Ying; Lin, Cheng-Wei; Shiao, Hui-Yi; Ho, Yun-Lung; Reddy, Randheer; Lin, Wen-Hsing; Chen, Chun-Hwa; Peng, Yi-Hui; Leou, Jiun-Shyang; Lien, Tzu-Wen; Huang, Chin-Ting; Fang, Ming-Yu; Wu, Szu-Huei; Wu, Jian-Sung; Chittimalla, Santhosh Kumar; Song, Jen-Shin; Hsu, John T-A; Wu, Su-Ying; Liao, Chun-Chen; Chao, Yu-Sheng; Hsieh, Hsing-Pang

2010-07-08

A focused library of furanopyrimidine (350 compounds) was rapidly synthesized in parallel reactors and in situ screened for Aurora and epidermal growth factor receptor (EGFR) kinase activity, leading to the identification of some interesting hits. On the basis of structural biology observations, the hit 1a was modified to better fit the back pocket, producing the potent Aurora inhibitor 3 with submicromolar antiproliferative activity in HCT-116 colon cancer cell line. On the basis of docking studies with EGFR hit 1s, introduction of acrylamide Michael acceptor group led to 8, which inhibited both the wild and mutant EGFR kinase and also showed antiproliferative activity in HCC827 lung cancer cell line. Furthermore, the X-ray cocrystal study of 3 and 8 in complex with Aurora and EGFR, respectively, confirmed their hypothesized binding modes. Library construction, in situ screening, and structure-based drug design (SBDD) strategy described here could be applied for the lead identification of other kinases.

Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

PubMed

McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

2016-01-01

The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors.

The novel HSP90 inhibitor NVP-AUY922 shows synergistic anti-leukemic activity with cytarabine in vivo.

PubMed

Wendel, Torunn; Zhen, Yan; Suo, Zenhe; Bruheim, Skjalg; Wiedlocha, Antoni

2016-01-15

HSP90 is a molecular chaperone essential for stability, activity and intracellular sorting of many proteins, including oncoproteins, such as tyrosine kinases, transcription factors and cell cycle regulatory proteins. Therefore, inhibitors of HSP90 are being investigated for their potential as anti-cancer drugs. Here we show that the HSP90 inhibitor NVP-AUY922 induced degradation of the fusion oncoprotein FOP2-FGFR1 in a human acute myeloid leukemia (AML) cell line, KG-1a. Concordantly, downstream signaling cascades, such as STAT1, STAT3 and PLCγ were abrogated. At concentrations that caused FOP2-FGFR1 degradation and signaling abrogation, NVP-AUY922 treatment caused significant cell death and inhibition of proliferation of KG-1a cells in vitro. In an animal model for AML, NVP-AUY922 administrated alone showed no anti-leukemic activity. However, when NVP-AUY922 was administered in combination with cytarabine, the two compounds showed significant synergistic anti-leukemic activity in vivo. Thus NVP-AUY922 and cytarabine combination therapy might be a prospective strategy for AML treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

PubMed

De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

2005-05-01

Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?inhibitor activity with respect to soy flour was 8.1%, 3.3% and 1% for hydrolysate, F(1-10)E and F(0.5-3), respectively. Both fractions presented high nutritive quality and reduced or null allergenicity. The trypsin inhibitor activity decreased along processing and could be a useful indicator for production of hypoallergenic proteins.

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

PubMed Central

2015-01-01

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors. PMID:25075558

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

PubMed

Cruz, Ana C B; Massena, Fábio S; Migliolo, Ludovico; Macedo, Leonardo L P; Monteiro, Norberto K V; Oliveira, Adeliana S; Macedo, Francisco P; Uchoa, Adriana F; Grossi de Sá, Maria F; Vasconcelos, Ilka M; Murad, Andre M; Franco, Octavio L; Santos, Elizeu A

2013-09-01

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

PubMed

Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

2016-09-15

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. Copyright © 2016. Published by Elsevier B.V.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

PubMed

Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

GSK-3β inhibitors suppressed neuroinflammation in rat cortex by activating autophagy in ischemic brain injury.

PubMed

Zhou, Xiaogang; Zhou, Jian; Li, Xilei; Guo, Chang'an; Fang, Taolin; Chen, Zhengrong

2011-07-29

Previous studies have shown that GSK-3β inhibitor could reduce infarct volume after ischemia brain injury. However, the underlying mechanisms of GSK-3β inhibitor involving neuroprotection remain poorly understood. In the present study, we demonstrated that GSK-3β inhibitor suppressed insult-induced neuroinflammation in rat cortex by increasing autophagy activation in ischemic injury. Male rats were subjected to pMCAO (permanent middle cerebral artery occlusion) followed by treating with SB216763, a GSK-3β inhibitor. We found that insult-induced inflammatory response was significantly decreased by intraperitoneal infusion of SB216763 in rat cortex. A higher level of autophagy was also detected after SB216763 treatment. In the cultured primary microglia, SB216763 activated autophagy and suppressed inflammatory response. Importantly, inhibition of autophagy by Beclin1-siRNA increased inflammatory response in the SB216763-treated microglia. These data suggest that GSK-3β inhibitor suppressed neuroinflammation by activating autophagy after ischemic brain injury, thus offering a new target for prevention of ischemic brain injury. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2006-01-01

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

Novel factor Xa inhibitors: a patent review.

PubMed

de Candia, Modesto; Lopopolo, Gianfranco; Altomare, Cosimo

2009-11-01

New oral anticoagulants with favorable safety profiles and fixed doses are required for the management of thromboembolism and stroke prevention in patients with atrial fibrillation. Among them, fXa inhibitors (the so-called xabans) are attractive options that can overcome limitations (e.g., bleeding) of the current oral antithrombotic therapy. The rational design of small-molecule direct fXa inhibitors, whose importance is testified by the growing number of publications and patents recently registered, has been fully supported by the X-ray crystallography of enzyme-ligand complexes. Pubmed, SciFinder Scholar, ISI web of knowledge(SM), http://ep.espacenet.com/ and Google websites were used as the main sources for literature retrieving, and > 100 patents filed between 2006 and April 2009, reviewed and discussed herein, highlight the variety among the P1 and P4 moieties on suitable scaffolds. The replacement of the benzamidine P1 moiety, which characterizes the first generation, with less basic bioisosteric or nonpolar neutral P1 groups led to the disclosure of numerous fXa inhibitors with high potency, selectivity and oral bioavailability. Novel selective fXa inhibitors with stable pharmacokinetics, better therapeutic windows and ease-of-use than the existing anticoagulants are currently under advanced stage clinical trials. Available data from Phase II and Phase III studies reflect the drive towards fXa inhibitors as potentially more effective and safer antithrombotic drugs. Their development is expected to address two major needs for anticoagulation, namely safety and ease-of-use, and to significantly affect the anticoagulant market.

Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein.

PubMed

Tsai, Ting-Yueh; Yeh, Teng-Kuang; Chen, Xin; Hsu, Tsu; Jao, Yu-Chen; Huang, Chih-Hsiang; Song, Jen-Shin; Huang, Yu-Chen; Chien, Chia-Hui; Chiu, Jing-Huai; Yen, Shih-Chieh; Tang, Hung-Kuan; Chao, Yu-Sheng; Jiaang, Weir-Torn

2010-09-23

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II.

PubMed

De Winter, Hans; Breslin, Henry; Miskowski, Tamara; Kavash, Robert; Somers, Marijke

2005-04-01

A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure-activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.

PubMed

Mitchell, Joanne L; Lionikiene, Ausra S; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y; Mutch, Nicola J

2016-12-15

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP 70 , of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP 70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP 70 Indeed, complex formation between polyP 70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP 70 , highlighting the importance of the anion binding site. PolyP 70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP 70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP 70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP 70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. © 2016 by The American Society of Hematology.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

PubMed Central

Lionikiene, Ausra S.; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y.

2016-01-01

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70. Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. PMID:27694320

Up-stream events in the nuclear factor κB activation cascade in response to sparsely ionizing radiation

NASA Astrophysics Data System (ADS)

Hellweg, Christine E.; Langen, Britta; Klimow, Galina; Ruscher, Roland; Schmitz, Claudia; Baumstark-Khan, Christa; Reitz, Günther

2009-10-01

Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear anti-apoptotic transcription factor nuclear factor κB (NF-κB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527-530, 2005]. Studies with NF-κB inhibitors can map functional details of the NF-κB pathway and the influence of radiation-induced NF-κB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-κB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-κB SN50 were analyzed using HEK-pNF-κB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-κB activation by the cytokine tumor necrosis factor α. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-κB SN-50 could not reduce NF-κB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132

Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as Anticancer Agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bello, A.; Konforte, D; Poduch, E

2009-01-01

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro,more » 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.« less

Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C.

PubMed

Ghimire, Dibya; Timilsina, Uddhav; Srivastava, Tryambak Pratap; Gaur, Ritu

2017-03-02

HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics.

Dual activities of ritanserin and R59022 as DGKα inhibitors and serotonin receptor antagonists.

PubMed

Boroda, Salome; Niccum, Maria; Raje, Vidisha; Purow, Benjamin W; Harris, Thurl E

2017-01-01

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzyme's affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.

PubMed

Lin, Su-Chang; Chung, Jee Y; Lamothe, Betty; Rajashankar, Kanagalaghatta; Lu, Miao; Lo, Yu-Chih; Lam, Amy Y; Darnay, Bryant G; Wu, Hao

2008-02-15

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.

PubMed

Coppola, Julia M; Hamilton, Christin A; Bhojani, Mahaveer S; Larsen, Martha J; Ross, Brian D; Rehemtulla, Alnawaz

2007-05-01

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

PubMed Central

Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

2007-01-01

Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

Delayed Treatment Acceleration in Patients with Rheumatoid Arthritis Who Have Inadequate Response to Initial Tumor Necrosis Factor Inhibitors: Data from the Corrona Registry

PubMed Central

Pappas, Dimitrios A.; Gerber, Robert A.; Litman, Heather J.; Gruben, David; Geier, Jamie; Hua, Winnie D.; Chen, Connie; Li, Youfu; Kremer, Joel M.; Andrews, John S.; Bourret, Jeffrey A.

2018-01-01

Background The implementation of treat-to-target principles in rheumatoid arthritis (RA) has not been fully investigated in patients with inadequate response to tumor necrosis factor (TNF) inhibitor treatment. Objectives To evaluate the prevalence of an inadequate response to initial TNF inhibitor treatment at 6 and 12 months among patients with RA in a real-world patient registry, as well as the delay in therapy adjustment and its impact on disease activity and patient-reported outcome (PRO) measures. Methods This analysis is based on data of patients with moderate or severe disease activity (Clinical Disease Activity Index [CDAI] score >10) who were included in the Consortium of Rheumatology Researchers of North America (Corrona) RA registry, a prospective, observational database. The patients had never received treatment with a biologic disease-modifying antirheumatic drug (DMARD) and had initiated treatment with a TNF inhibitor (adalimumab, certolizumab pegol, etanercept, golimumab, or infliximab) between October 2001 and December 2014. We evaluated treatment response (CDAI score ≤10), select PRO measures, and treatment changes at 6 months. Patients who had an inadequate response to TNF inhibitor therapy at 6 months and continued to use their initial TNF inhibitor were evaluated again at 12 months. Results This retrospective analysis included 2282 patients. At 6 months, 1732 (75.9%) of the patients continued to use their initial TNF inhibitor; of these, 803 (46.4%) patients had an inadequate response to treatment. Of the 803 patients who had an inadequate response at 6 months, 488 (60.8%) continued their initial treatment at 12 months. Of these 488 patients, 315 (64.5%) had an inadequate response at 12 months, and 173 (35.5%) had a response. Numerically greater improvements in all PRO measures were observed for patients who responded to therapy compared with patients with an inadequate response. Conclusions In this real-world analysis of data from the Corrona

Epistatic effect of plasminogen activator inhibitor 1 and beta-fibrinogen genes on risk of glomerular microthrombosis in lupus nephritis: interaction with environmental/clinical factors.

PubMed

Gong, Rujun; Liu, Zhihong; Li, Leishi

2007-05-01

Glomerular microthrombosis (GMT) is not uncommon in lupus nephritis and has been associated with active renal injury and progressive kidney destruction. We undertook this study to determine whether genetic variations of hemostasis factors, such as plasminogen activator inhibitor 1 (PAI-1) and fibrinogen, affect the risk of GMT. A cross-sectional cohort of 101 lupus nephritis patients with or without GMT was genotyped for PAI-1 -675 4G/5G and beta-fibrinogen (FGB) -455 G/A gene polymorphisms and analyzed. PAI-1 4G/4G homozygotes and FGB A allele carriers were both at increased risk for GMT. When the data were stratified for both gene polymorphisms, an epistatic effect was detected. The PAI-1 4G/4G genotype was found to predispose to GMT not equally in all lupus nephritis patients, but only in FGB A allele carriers. Likewise, the association between the FGB A allele and GMT was restricted to lupus nephritis patients homozygous for the PAI-1 4G allele. This epistatic effect was revalidated by the multifactor dimensionality reduction (MDR) analysis and further assessed by incorporating a variety of environmental and clinical factors into the MDR analysis. The most parsimonious model that had a cross-validation consistency of 100% included joint effects of PAI-1 and FGB gene polymorphisms and anticardiolipin antibody (aCL) status and yielded the best prediction of GMT, with 66.6% accuracy. Our findings suggest that risk of GMT in lupus nephritis is attributable, at least in part, to an epistatic effect of PAI-1 and FGB genes, likely via an interaction with environmental/clinical factors, such as aCL.

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak

Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

DOE PAGES

Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak; ...

2014-07-17

Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

Improved antitumor activity of immunotherapy with BRAF and MEK inhibitors in BRAFV600E melanoma

PubMed Central

Hu-Lieskovan, Siwen; Mok, Stephen; Moreno, Blanca Homet; Tsoi, Jennifer; Faja, Lidia Robert; Goedert, Lucas; Pinheiro, Elaine M.; Koya, Richard C.; Graeber, Thomas; Comin-Anduix, Begoña; Ribas, Antoni

2016-01-01

Combining immunotherapy and BRAF targeted therapy may result in improved antitumor activity with the high response rates of targeted therapy and the durability of responses with immunotherapy. However, the first clinical trial testing the combination of the BRAF inhibitor vemurafenib and the CTLA-4 antibody ipilimumab was terminated early due to substantial liver toxicities. MEK inhibitors can potentiate the MAPK inhibition in BRAF mutant cells, while potentially alleviating the unwanted paradoxical MAPK activation in BRAF wild type cells that lead to side effects when using BRAF inhibitors alone. However, there is the concern of MEK inhibitors being detrimental to T cell functionality. Using a mouse model of syngeneic BRAFV600E driven melanoma, we tested whether addition of the MEK inhibitor trametinib would enhance the antitumor activity of combined immunotherapy with the BRAF inhibitor dabrafenib. Combination of dabrafenib and trametinib with pmel-1 adoptive cell transfer (ACT) showed complete tumor regression, increased T cell infiltration into tumors and improved in vivo cytotoxicity. Single agent dabrafenib increased tumor-associated macrophages and T regulatory cells (Tregs) in tumors, which decreased with the addition of trametinib. The triple combination therapy resulted in increased melanosomal antigen and MHC expression, and global immune-related gene up-regulation. Given the up-regulation of PD-L1 seen with dabrafenib and/or trametinib combined with antigen-specific ACT, we tested combination of dabrafenib, trametinib with anti-PD1 therapy in SM1 tumors, and observed superior anti-tumor effect. Our findings support the testing of triple combination therapy of BRAF and MEK inhibitors with immunotherapy in patients with BRAFV600E mutant metastatic melanoma. PMID:25787767

C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

PubMed

Papareddy, Praveen; Kalle, Martina; Kasetty, Gopinath; Mörgelin, Matthias; Rydengård, Victoria; Albiger, Barbara; Lundqvist, Katarina; Malmsten, Martin; Schmidtchen, Artur

2010-09-03

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.

Melasma treatment: A novel approach using a topical agent that contains an anti-estrogen and a vascular endothelial growth factor inhibitor.

PubMed

Cohen, Philip R

2017-04-01

Melasma is an acquired disorder of pigmentation that presents with asymptomatic symmetric darkening of the face. The pathogenesis of this condition is multifactorial and influenced by several factors including female sex hormones, genetic predisposition and ultraviolet light exposure. The management of melasma is usually directed at more than one of the causative etiologic factors and often incorporates a combination of topical agents, with or without the addition of physical modalities. Estrogen and angiogenesis are significant factors in the etiology of melasma. A useful addition to the therapeutic armentarium for treating melasma would include a topical agent that could effect both of these causative factors. Specifically, a topical preparation consisting of an anti-estrogen and a vascular endothelial growth factor inhibitor would accomplish this goal. Suitable candidates that target estrogen receptors and vascular endothelial growth factor are currently used in medical oncology as systemic antineoplastic agents. The anti-estrogen could be either a selective estrogen receptor modulator (such as tamoxifen or raloxifene) or an aromatase inhibitor (such as anastrozole or letrozole or exemestane). The vascular endothelial growth factor inhibitor would be bevacizumab. In conclusion, a novel-topically administered-therapy for melasma would combine an anti-estrogen and a vascular endothelial growth factor inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhanced venous thrombus resolution in plasminogen activator inhibitor type-2 deficient mice.

PubMed

Siefert, S A; Chabasse, C; Mukhopadhyay, S; Hoofnagle, M H; Strickland, D K; Sarkar, R; Antalis, T M

2014-10-01

The resolution of deep vein thrombosis requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. To investigate the role of PAI-2 in venous thrombus formation and resolution. Venous thrombus resolution was compared in wild-type C57BL/6, PAI-2(-/-) , and PAI-1(-/-) mice using the stasis model of deep vein thrombosis. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. We found that the absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2(-/-) mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2-deficient thrombi had increased levels of the neutrophil chemoattractant CXCL2, which was associated with early enhanced neutrophil recruitment. These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. © 2014 International Society on Thrombosis and Haemostasis.

Evidence that the modulation of membrane-associated protein kinase C activity by an endogenous inhibitor plays a role in N1E-115 murine neuroblastoma cell differentiation.

PubMed

Chakravarthy, B R; Wong, J; Durkin, J P

1995-10-01

Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.

Design, synthesis and structure-activity relationship evaluation of novel LpxC inhibitors as Gram-negative antibacterial agents.

PubMed

Ding, Shi; Dai, Rui-Yang; Wang, Wen-Ke; Cao, Qiao; Lan, Le-Fu; Zhou, Xian-Li; Yang, Yu-She

2018-01-15

LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly against Gram-negative bacteria infections. To develop novel LpxC inhibitors with good antibacterial activities and biological metabolism, we summarized the basic skeleton of reported LpxC inhibitors, designed and synthesized several series of compounds and tested their antibacterial activities against Escherichial coli and Pseudomonas aeruginosa in vitro. Structure-activity relationships have been discussed in this article. The metabolism stability of YDL-2, YDL-5, YDL-8, YDL-14, YDL-20-YDL-23 have been evaluated in liver microsomes, which indicated that the 2-amino isopropyl group may be a preferred structure than the 2-hydroxy ethyl group in the design of LpxC inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Condensed Tannins from Longan Bark as Inhibitor of Tyrosinase: Structure, Activity, and Mechanism.

PubMed

Chai, Wei-Ming; Huang, Qian; Lin, Mei-Zhen; Ou-Yang, Chong; Huang, Wen-Yang; Wang, Ying-Xia; Xu, Kai-Li; Feng, Hui-Ling

2018-01-31

In this study, the content, structure, antityrosinase activity, and mechanism of longan bark condensed tannins were evaluated. The findings obtained from mass spectrometry demonstrated that longan bark condensed tannins were mixtures of procyanidins, propelargonidins, prodelphinidins, and their acyl derivatives (galloyl and p-hydroxybenzoate). The enzyme analysis indicated that these mixtures were efficient, reversible, and mixed (competitive is dominant) inhibitor of tyrosinase. What's more, the mixtures showed good inhibitions on proliferation, intracellular enzyme activity and melanogenesis of mouse melanoma cells (B 16 ). From molecular docking, the results showed the interactions between inhibitors and tyrosinase were driven by hydrogen bond, electrostatic, and hydrophobic interactions. In addition, high levels of total phenolic and extractable condensed tannins suggested that longan bark might be a good source of tyrosinase inhibitor. This study would offer theoretical basis for the development of longan bark condensed tannins as novel food preservatives and medicines of skin diseases.

Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations.

PubMed

Yu, Zhe; Ma, Yu-chi; Ai, Jing; Chen, Dan-qi; Zhao, Dong-mei; Wang, Xin; Chen, Yue-lei; Geng, Mei-yu; Xiong, Bing; Cheng, Mao-sheng; Shen, Jing-Kang

2013-11-01

To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π-π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.