Sample records for dynamin inhibitor inhibits

  1. Some selective serotonin reuptake inhibitors inhibit dynamin I guanosine triphosphatase (GTPase). (United States)

    Otomo, Masahiro; Takahashi, Kiyofumi; Miyoshi, Hiroshi; Osada, Kenichi; Nakashima, Hideki; Yamaguchi, Noboru


    Neuronal dynamin I plays a critical role in the recycling of synaptic vesicles, and thus in nervous system function. We expressed and purified dynamin I to explore potentially clinically useful endocytosis inhibitors and to examine the mechanism of their action. We estimated the IC(50) of nineteen psychotropic drugs for dynamin I. The IC(50) values of two selective serotonin reuptake inhibitors (sertraline and fluvoxamine) were 7.3+/-1.0 and 14.7+/-1.6 microM, respectively. Kinetic analyses revealed that fluvoxamine is a noncompetitive inhibitor of dynamin I guanosine triphosphatase (GTPase) with respect to guanosine 5'-triphosphate (GTP) and a competitive inhibitor with respect to L-phosphatidylserine (PS). Fluvoxamine may compete with PS for binding to the pleckstrin homology domain of dynamin I. On the other hand, sertraline was a mixed type inhibitor with respect to both GTP and PS. Our results indicate that sertraline and fluvoxamine may regulate the transportation of neurotransmitters by modulating synaptic vesicle endocytosis via the inhibition of dynamin I GTPase.

  2. Dynasore, a dynamin inhibitor, inhibits Trypanosoma cruzi entry into peritoneal macrophages.

    Directory of Open Access Journals (Sweden)

    Emile S Barrias

    Full Text Available BACKGROUND: Trypanosoma cruzi is an intracellular parasite that, like some other intracellular pathogens, targets specific proteins of the host cell vesicular transport machinery, leading to a modulation of host cell processes that results in the generation of unique phagosomes. In mammalian cells, several molecules have been identified that selectively regulate the formation of endocytic transport vesicles and the fusion of such vesicles with appropriate acceptor membranes. Among these, the GTPase dynamin plays an important role in clathrin-mediated endocytosis, and it was recently found that dynamin can participate in a phagocytic process. METHODOLOGY/PRINCIPAL FINDINGS: We used a compound called dynasore that has the ability to block the GTPase activity of dynamin. Dynasore acts as a potent inhibitor of endocytic pathways by blocking coated vesicle formation within seconds of its addition. Here, we investigated whether dynamin is involved in the entry process of T. cruzi in phagocytic and non-phagocytic cells by using dynasore. In this aim, peritoneal macrophages and LLC-MK2 cells were treated with increasing concentrations of dynasore before interaction with trypomastigotes, amastigotes or epimastigotes. We observed that, in both cell lines, the parasite internalization was drastically diminished (by greater than 90% in LLC-MK2 cells and 70% in peritoneal macrophages when we used 100 microM dynasore. The T. cruzi adhesion index, however, was unaffected in either cell line. Analyzing these interactions by scanning electron microscopy and comparing peritoneal macrophages to LLC-MK2 cells revealed differences in the stage at which cell entry was blocked. In LLC-MK2 cells, this blockade is observed earlier than it is in peritoneal macrophages. In LLC-MK2 cells, the parasites were only associated with cellular microvilli, whereas in peritoneal macrophages, trypomastigotes were not completely engulfed by a host cell plasma membrane. CONCLUSIONS

  3. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  4. Suppression of dynamin GTPase activity by sertraline leads to inhibition of dynamin-dependent endocytosis. (United States)

    Takahashi, Kiyofumi; Miyoshi, Hiroshi; Otomo, Masahiro; Osada, Kenichi; Yamaguchi, Noboru; Nakashima, Hideki


    Dynamin (Dyn) 1 plays a role in recycling of synaptic vesicles, and thus in nervous system function. We previously showed that sertraline, a selective serotonin reuptake inhibitor (SSRI), is a mixed-type inhibitor of Dyn 1 with respect to both GTP and L-alpha-phosphatidyl-L-serine (PS) in vitro, and we suggested that it may regulate the neurotransmitter transport by modulating synaptic vesicle endocytosis via inhibition of Dyn 1 GTPase. Here, we investigated the effect of sertraline on endocytosis of marker proteins in human neuroblastoma SH-Sy5Y cells and HeLa cells. Sertraline inhibited endocytosis in both cell lines. Western blotting showed that SH-Sy5Y expresses Dyn 1 and Dyn 2, while HeLa expresses only Dyn 2. GTPase assay showed that sertraline inhibited Dyn 2 as well as Dyn 1. Therefore, the effect of sertraline on endocytosis was mediated by Dyn 2, at least in HeLa cells, as well as by Dyn 1 in cell lines that express it. Moreover, the inhibition mechanism of transferrin (Tf) uptake by sertraline differed from that in cells expressing Dyn 1 K44A, a GTP binding-defective variant, and sertraline did not interfere with the interaction between Dyn 1 and PS-liposomes.

  5. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. (United States)

    McCluskey, Adam; Daniel, James A; Hadzic, Gordana; Chau, Ngoc; Clayton, Emma L; Mariana, Anna; Whiting, Ainslie; Gorgani, Nick N; Lloyd, Jonathan; Quan, Annie; Moshkanbaryans, Lia; Krishnan, Sai; Perera, Swetha; Chircop, Megan; von Kleist, Lisa; McGeachie, Andrew B; Howes, Mark T; Parton, Robert G; Campbell, Michael; Sakoff, Jennette A; Wang, Xuefeng; Sun, Jian-Yuan; Robertson, Mark J; Deane, Fiona M; Nguyen, Tam H; Meunier, Frederic A; Cousin, Michael A; Robinson, Phillip J


    Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ~ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

  6. Surface expression and limited proteolysis of ADAM10 are increased by a dominant negative inhibitor of dynamin

    Directory of Open Access Journals (Sweden)

    Slack Barbara E


    Full Text Available Abstract Background The amyloid precursor protein (APP is cleaved by β- and γ-secretases to generate toxic amyloid β (Aβ peptides. Alternatively, α-secretases cleave APP within the Aβ domain, precluding Aβ formation and releasing the soluble ectodomain, sAPPα. We previously showed that inhibition of the GTPase dynamin reduced APP internalization and increased release of sAPPα, apparently by prolonging the interaction between APP and α-secretases at the plasma membrane. This was accompanied by a reduction in Aβ generation. In the present study, we investigated whether surface expression of the α-secretase ADAM (a disintegrin and metalloprotease10 is also regulated by dynamin-dependent endocytosis. Results Transfection of human embryonic kidney (HEK cells stably expressing M3 muscarinic receptors with a dominant negative dynamin I mutant (dyn I K44A, increased surface expression of both immature, and mature, catalytically active forms of co-expressed ADAM10. Surface levels of ADAM10 were unaffected by activation of protein kinase C (PKC or M3 receptors, indicating that receptor-coupled shedding of the ADAM substrate APP is unlikely to be mediated by inhibition of ADAM10 endocytosis in this cell line. Dyn I K44A strongly increased the formation of a C-terminal fragment of ADAM10, consistent with earlier reports that the ADAM10 ectodomain is itself a target for sheddases. The abundance of this fragment was increased in the presence of a γ-secretase inhibitor, but was not affected by M3 receptor activation. The dynamin mutant did not affect the distribution of ADAM10 and its C-terminal fragment between raft and non-raft membrane compartments. Conclusions Surface expression and limited proteolysis of ADAM10 are regulated by dynamin-dependent endocytosis, but are unaffected by activation of signaling pathways that upregulate shedding of ADAM substrates such as APP. Modulation of ADAM10 internalization could affect cellular behavior in two

  7. Osteoblast differentiation and migration are regulated by dynamin GTPase activity. (United States)

    Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W; Bruzzaniti, Angela


    Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation.

  8. Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation. (United States)

    Din, Shabana; Mason, Matthew; Völkers, Mirko; Johnson, Bevan; Cottage, Christopher T; Wang, Zeping; Joyo, Anya Y; Quijada, Pearl; Erhardt, Peter; Magnuson, Nancy S; Konstandin, Mathias H; Sussman, Mark A


    Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

  9. Dynamin 1 is required for memory formation.

    Directory of Open Access Journals (Sweden)

    Mauro Fà

    Full Text Available Dynamin 1-3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity.

  10. Dynamin Binding Protein (Tuba) Deficiency Inhibits Ciliogenesis and Nephrogenesis in Vitro and in Vivo. (United States)

    Baek, Jeong-In; Kwon, Sang-Ho; Zuo, Xiaofeng; Choi, Soo Young; Kim, Seok-Hyung; Lipschutz, Joshua H


    Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity.

  11. Kalirin12 interacts with dynamin

    Directory of Open Access Journals (Sweden)

    Mains Richard E


    Full Text Available Abstract Background Guanine nucleotide exchange factors (GEFs and their target Rho GTPases regulate cytoskeletal changes and membrane trafficking. Dynamin, a large force-generating GTPase, plays an essential role in membrane tubulation and fission in cells. Kalirin12, a neuronal RhoGEF, is found in growth cones early in development and in dendritic spines later in development. Results The IgFn domain of Kalirin12, not present in other Kalirin isoforms, binds dynamin1 and dynamin2. An inactivating mutation in the GTPase domain of dynamin diminishes this interaction and the isolated GTPase domain of dynamin retains the ability to bind Kalirin12. Co-immunoprecipitation demonstrates an interaction of Kalirin12 and dynamin2 in embryonic brain. Purified recombinant Kalirin-IgFn domain inhibits the ability of purified rat brain dynamin to oligomerize in response to the presence of liposomes containing phosphatidylinositol-4,5-bisphosphate. Consistent with this, expression of exogenous Kalirin12 or its IgFn domain in PC12 cells disrupts clathrin-mediated transferrin endocytosis. Similarly, expression of exogenous Kalirin12 disrupts transferrin endocytosis in cortical neurons. Expression of Kalirin7, a shorter isoform which lacks the IgFn domain, was previously shown to inhibit clathrin-mediated endocytosis; the GTPase domain of dynamin does not interact with Kalirin7. Conclusion Kalirin12 may play a role in coordinating Rho GTPase-mediated changes in the actin cytoskeleton with dynamin-mediated changes in membrane trafficking.

  12. Dynamin-related protein inhibitor downregulates reactive oxygen species levels to indirectly suppress high glucose-induced hyperproliferation of vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Maimaitijiang, Alimujiang; Zhuang, Xinyu; Jiang, Xiaofei; Li, Yong, E-mail:


    Hyperproliferation of vascular smooth muscle cells is a pathogenic mechanism common in diabetic vascular complications and is a putatively important therapeutic target. This study investigated multiple levels of biology, including cellular and organellar changes, as well as perturbations in protein synthesis and morphology. Quantitative and qualitative analysis was utilized to assess the effect of mitochondrial dynamic changes and reactive oxygen species(ROS) levels on high-glucose-induced hyperproliferation of vascular smooth muscle cells. The data demonstrated that the mitochondrial fission inhibitor Mdivi-1 and downregulation of ROS levels both effectively inhibited the high-glucose-induced hyperproliferation of vascular smooth muscle cells. Downregulation of ROS levels played a more direct role and ROS levels were also regulated by mitochondrial dynamics. Increased ROS levels induced excessive mitochondrial fission through dynamin-related protein (Drp 1), while Mdivi-1 suppressed the sensitivity of Drp1 to ROS levels, thus inhibiting excessive mitochondrial fission under high-glucose conditions. This study is the first to propose that mitochondrial dynamic changes and ROS levels interact with each other and regulate high-glucose-induced hyperproliferation of vascular smooth muscle cells. This finding provides novel ideas in understanding the pathogenesis of diabetic vascular remodeling and intervention. - Highlights: • Mdivi-1 inhibits VSMC proliferation by lowering ROS level in high-glucose condition. • ROS may be able to induce mitochondrial fission through Drp1 regulation. • Mdivi-1 can suppress the sensitivity of Drp1 to ROS.

  13. Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent (United States)

    Hsu, Mei-Ju; Rixon, Frazer J.; Knebel-Mörsdorf, Dagmar


    Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol. PMID:22022400

  14. Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.

    Directory of Open Access Journals (Sweden)

    Elena Rahn

    Full Text Available Herpes simplex virus type 1 (HSV-1 can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s. Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.

  15. Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression

    Directory of Open Access Journals (Sweden)

    Braithwaite Antony W


    Full Text Available Abstract Background The aim of both classical (e.g. taxol and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs, which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. Results We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. Conclusion Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

  16. Dynamin 1 regulates amyloid generation through modulation of BACE-1.

    Directory of Open Access Journals (Sweden)

    Li Zhu

    Full Text Available BACKGROUND: Several lines of investigation support the notion that endocytosis is crucial for Alzheimer's disease (AD pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1 distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1 in Aβ biogenesis. PRINCIPAL FINDINGS: We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF. Moreover, dyn1 knockdown (KD leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α-secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT or PS1 N-terminal fragment (NTF at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1(-/- primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts. CONCLUSIONS: In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its

  17. The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Girard

    Full Text Available Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC within the endolysosomal network. The measure of cholesterol esters (CE further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2, 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR, and low-density lipoprotein receptor (LDLR. The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

  18. Insulin promotes Rip11 accumulation at the plasma membrane by inhibiting a dynamin- and PI3-kinase-dependent, but Akt-independent, internalisation event. (United States)

    Boal, Frédéric; Hodgson, Lorna R; Reed, Sam E; Yarwood, Sophie E; Just, Victoria J; Stephens, David J; McCaffrey, Mary W; Tavaré, Jeremy M


    Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis.

  19. Scale Inhibition of Green Inhibitor Polyepoxysuccinic Sodium

    Institute of Scientific and Technical Information of China (English)

    Feng Hui-xia; Wang Yi; Yu Shu-rong; Liang Bao-feng


    Polyepoxysuccinic acid (PESA) is the green water treatment agents recognized all over the world[1-3]. It is found that when PESA is used alone, it had good scale inhibition. PESA should be included in the category of green scale inhibitor.PESA is synthesized with maleicanhydride in the presence of catalysts. The effect on scale-in-hibiting property of the product from amount and feed times of catalyst, the reaction temperature, the reaction time were investigated. The optimum reaction conditions are as follows:n(maleic anhydride):n(Ca(OH)2):n(NaOH)=1:0.05-0.2:0.5, reaction temperature 95C, reaction time 4h.In all the references about PESA, PESA is researched as a kind of highly effective scale inhibitor or chelate. In this paper, the performance of scale inhibition of PESA is evaluated by scale static inhibitor.The results are shown in Figture1.It is evident from our experimental data (Figture1) that when inhibition for CaCO3.With the increase of PESA dosage, scale inhibition increases. When dosage is more than 6mg/L, inhibition efficiency is over 50%. The formulas give scale inhibition efficiency more than 95% at 12mg/L of total dosage.

  20. Class III phosphoinositide 3-kinase/VPS34 and dynamin are critical for apical endocytic recycling. (United States)

    Carpentier, Sarah; N'Kuli, Francisca; Grieco, Giuseppina; Van Der Smissen, Patrick; Janssens, Virginie; Emonard, Hervé; Bilanges, Benoît; Vanhaesebroeck, Bart; Gaide Chevronnay, Héloïse P; Pierreux, Christophe E; Tyteca, Donatienne; Courtoy, Pierre J


    Recycling is a limiting step for receptor-mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan-class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP-2, causing surface down-regulation. GFP-(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002-treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin-GFP and dependence of dynamin-GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K-III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

  1. Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB


    King, Megan C.; Raposo, Graça; Lemmon, Mark A.


    Mx proteins form a subfamily of the dynamin-like GTPases, which have well established roles in cellular trafficking. Some Mx proteins (e.g., human MxA) have antiviral activity and are tightly regulated by type I IFNs. Others (e.g., human MxB) lack antiviral activity and are thought to have normal cellular functions that remain undefined. Consistent with this hypothesis, we report that MxB is expressed without IFN treatment. MxB seems to be exclusively extranuclear and is concentrated at the c...

  2. Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB. (United States)

    King, Megan C; Raposo, Graça; Lemmon, Mark A


    Mx proteins form a subfamily of the dynamin-like GTPases, which have well established roles in cellular trafficking. Some Mx proteins (e.g., human MxA) have antiviral activity and are tightly regulated by type I IFNs. Others (e.g., human MxB) lack antiviral activity and are thought to have normal cellular functions that remain undefined. Consistent with this hypothesis, we report that MxB is expressed without IFN treatment. MxB seems to be exclusively extranuclear and is concentrated at the cytoplasmic face of nuclear pores, suggesting a role in their regulation. We find that expression of dominant negative (GTPase-defective) MxB mutants efficiently blocks nuclear import and causes a delay in G(1)/S cell-cycle progression. MxB depletion using RNA interference (RNAi) leads to a similar cell-cycle defect but does not block nuclear import. MxB therefore seems not to be required for nuclear import per se but may instead regulate its efficiency and/or kinetics. These studies indicate an unexpected role for a dynamin-like protein in nucleocytoplasmic trafficking and suggest that a related function might be usurped by its antiviral relatives.

  3. Screening and identification of dynamin-1 interacting proteins in rat brain synaptosomes. (United States)

    Zhang, Ciliu; Omran, Ahmed Galal; He, Fang; Deng, Xiaolu; Wu, Lei; Peng, Jing; Yin, Fei


    Dynamin-1 is a multi-domain GTPase that is crucial for the fission stage of synaptic vesicle recycling and vesicle trafficking. In this study, we constructed prokaryotic expression plasmids for the four functional domains of dynamin-1, which are pGEX-4T-2-PH, pGEX-4T-2-PRD, pGEX-4T-2-GED and pGEX-4T-2-GTPase. Glutathione S-transferase pull-down, co-immunoprecipitation (co-IP), and liquid chromatography/mass spectrometry were used to screen and identify dynamin-1 interacting proteins in rat brain synaptosomes. We identified a set of 63 candidate protein interactions, including 36 proteins interacting with dynamin-1 C-terminal proline-rich domain (PRD), 14 with pleckstrin-homology domain (PH), 7 with GTPase effector domain (GED) and 6 with GTPase domain, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. We selected three previously unreported dynamin-1 interacting proteins to verify their interaction with dynamin-1 under native conditions. Using co-IP, we found that Rab GDP-dissociation inhibitor (Rab GDI) and chloride channel 3 (ClC-3) do interact with dynamin-1, but not with TUC-4b (the TOAD-64/Ulip/CRMP (TUC) family member). Those novel interactions detected in our study offer valuable insight into the protein-protein interacting network that could enhance our understanding of dynamin-1 mediated synaptic vesicle recycling.

  4. Dynamin- and clathrin-dependent endocytic pathway in unicellular eukaryote Paramecium. (United States)

    Wiejak, Jolanta; Surmacz, Liliana; Wyroba, Elzbieta


    The first evidence of dynamin presence and its colocalization with clathrin in the compartment involved in Paramecium receptor-mediated endocytosis is presented. We identified dynamin by cloning, Western blotting, and immunodetection in confocal and electron microscopy. The partial genes, which we have designated ParDyn1 and ParDyn2, are 1091 bp long, 90% identical to one another and encode the N-terminal and middle domains of Paramecium dynamin isoform 1 and isoform 2. The deduced amino acid sequences contain all three guanosine 5'-triphosphate (GTP)-binding motifs and show 67% homology to mammalian dynamins. Antibodies generated against the cloned GTPase domain revealed dynamin association with endosomes containing transferrin, the marker of receptor-mediated endocytosis. In Western blotting a strong immunoreactive polypeptide of approximately 116 kDa, which seems to be phosphorylated, was accompanied by a faint one of approximately 90 kDa in cytosolic fraction (S2). Dynamin level was correlated with internalization of transferrin and it was significantly decreased upon inhibition of this process. Immunogold labeling in electron microscopy revealed colocalization of dynamin and clathrin in coated pits and endocytic vesicles. Moreover, the polypeptide cross-reaction with 2 different antibodies against mammalian clathrin was identified by immunoblotting. These results indicate that dynamin- and clathrin-dependent pathway exists in this evolutionary ancient cell.

  5. Dynamin: characteristics, mechanism of action and function. (United States)

    Wiejak, Jolanta; Wyroba, Elzbieta


    Dynamin - a member of the GTP-ase protein family - is essential for many intracellular membrane trafficking events in multiple endocytic processes. The unique biochemical features of dynamin - especially its propensity to assemble - enable severing the nascent vesicles from the membrane. The mechanism of dynamin's action is still a subject of debate - whether it functions as a mechanochemical enzyme or a regulatory GTPase. The GTPase domain of dynamin contains three GTP-binding motifs. This domain is very conservative across the species, including that recently cloned by us in the unicellular eukaryote Paramecium. Dynamin interacts with a number of partners such as endophilin and proteins involved in coordination of endocytosis with motor molecules. A growing body of evidence indicates that dynamin and dynamin-related proteins are involved both in pathology and protection against human diseases. The most interesting are dynamin-like Mx proteins exhibiting antiviral activity.

  6. Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry▿ (United States)

    Hernaez, Bruno; Alonso, Covadonga


    African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na+/H+ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed. PMID:19939916

  7. Corrosion Inhibition of a Green Scale Inhibitor Polyepoxysuccinic Acid

    Institute of Scientific and Technical Information of China (English)

    Rong Chun XIONG; Qing ZHOU; Gang WEI


    The corrosion inhibition of a green scale inhibitor, polyepoxysuccinic acid (PESA) wasstudied based on dynamic tests. It is found that when PESA is used alone, it had good corrosioninhibition. So, PESA should be included in the category of corrosion inhibitors. It is not only akind of green scale inhibitor, but also a green corrosion inhibitor. The synergistic effect betweenPESA and Zn2+ or sodium gluconate is poor. However, the synergistic effect among PESA, Zn2+and sodium gluconate is excellent, and the corrosion inhibition efficiency for carbon steel is higherthan 99%. Further study of corrosion inhibition mechanism reveals that corrosion inhibition ofPESA is not affected by carboxyl group, but by the oxygen atom inserted The existence ofoxygen atom in PESA molecular structure makes it easy to form stable chelate with pentacyclicstructure.

  8. Dynamin like protein 1 participated in the hemoglobin uptake pathway of Plasmodium falciparum

    Institute of Scientific and Technical Information of China (English)

    ZHOU Hong-chang; GAO Yu-hui; ZHONG Xiang; WANG Heng


    Background During the blood stage of malaria infection, parasites internalize in the host red blood cells and degrade massive amounts of hemoglobin for their development. Although the morphology of the parasite's hemoglobin uptake pathway has been clearly observed, little has been known about its molecular mechanisms. Methods The recombinant proteins from Plasmodium falciparum, dynamin like protein 1 (PfDYN1) and 2 (PfDYN2) GTPase domain, were expressed in E .coli and showed GTPase activity. By using a dynamin inhibitor, dynasore, we demonstrated the involvement of PfDYN1 in the hemoglobin uptake pathway. Results The GTPase activity of the two recombinant proteins was inhibited by dynasore in vitro. Treatment of parasite cultures with 80 μmol/L dynasore at the ring and early trophozoite stage resulted in substantial inhibition of parasite growth and in an obvious decline of hemoglobin quantum. Furthermore, reduced intraceliular hemozoin accumulation and decreased uptake of the FITC-dextran were also observed, together with distinctive changes in the ultrastructure of parasites after the dynasore treatment. Conclusions Our results show that PfDYN1 plays an important role in the hemoglobin uptake pathway of P. Falciparum and suggest its possibility of being a novel target for malaria chemotherapy.

  9. Protection from latent inhibition provided by a conditioned inhibitor. (United States)

    McConnell, Bridget L; Wheeler, Daniel S; Urcelay, Gonzalo P; Miller, Ralph R


    Two conditioned suppression experiments with rats investigated the influence on latent inhibition of compounding a Pavlovian conditioned inhibitor with the target cue during preexposure treatment. Results were compared with those of subjects that received conventional latent inhibition training, no preexposure, or preexposure to the target cue in compound with a neutral stimulus. In Experiment 1, greater attenuation of the latent inhibition effect was observed in subjects that received target preexposure in compound with a Pavlovian conditioned inhibitor relative to subjects that received preexposure with a neutral stimulus or to the target alone. In Experiment 2, this protection from latent inhibition was attenuated if the excitor that was used to train the conditioned inhibitor was extinguished between preexposure and target training. The results are consistent with an account offered by the extended comparator hypothesis.

  10. Protection from Latent Inhibition Provided by a Conditioned Inhibitor


    McConnell, Bridget L.; Wheeler, Daniel S.; Urcelay, Gonzalo P; Miller, Ralph R.


    Two conditioned suppression experiments with rats investigated the influence on latent inhibition of compounding a Pavlovian conditioned inhibitor with the target cue during preexposure treatment. Results were compared to subjects that received conventional latent inhibition training, no preexposure, or preexposure to the target cue in compound with a neutral stimulus. In Experiment 1, greater attenuation of the latent inhibition effect was observed in subjects that received target preexposur...

  11. Inhibition of matrix metalloproteinase-2 by PARP inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D. [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada); Pacher, Pal [National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Bethesda, MD (United States); Schulz, Richard, E-mail: [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada)


    Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC{sub 50} values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

  12. Inhibition of human immunodeficiency virus type-1 by cdk inhibitors

    Directory of Open Access Journals (Sweden)

    Kehn-Hall Kylene


    Full Text Available Abstract Current therapy for human immunodeficiency virus (HIV-1 infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202 in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs.

  13. Proton pump inhibitors inhibit pancreatic secretion

    DEFF Research Database (Denmark)

    Wang, Jing; Barbuskaite, Dagne; Tozzi, Marco


    +/K+-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKβ subunits (ATP4A; ATP4B) and non-gastric HKα2 subunits (ATP12A) of H+/K+-ATPases are expressed in human pancreatic cells. Pumps have similar...... of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO3-, pancreas also secretes K+. In conclusion, this study calls for a revision of the basic model for HCO3- secretion. We propose that proton transport is driving secretion, and that in addition...

  14. μ-Opioid Agonist Inhibition of κ-Opioid Receptor-Stimulated Extracellular Signal-Regulated Kinase Phosphorylation Is Dynamin-Dependent in C6 Glioma Cells


    Bohn, Laura M.; Belcheva, Mariana M.; Coscia, Carmine J.


    In previous studies we found that μ-opioids, acting via μ-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the κ-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with μ-opioid agonists for 1 h results in the inhibition of κ-opioid mitogenic signaling. The μ-selective agonist endomorphin-1 attenuates κ-opioid-stimu...

  15. Reduction of dynamin 1 in the hippocampus of aged mice is associated with the decline in hippocampal‑dependent memory. (United States)

    Yoo, Dae Young; Jung, Hyo Young; Kim, Jong Whi; Yim, Hee Sun; Kim, Dae Won; Nam, Hajin; Suh, Jun Gyo; Choi, Jung Hoon; Won, Moo-Ho; Yoon, Yeo Sung; Hwang, In Koo


    Dynamin 1 is a known synaptic protein, which has is key in the presynaptic regulation of endocytosis. The present study investigated the association between age and the observed changes in Morris water maze performance, and immunoreactivity and protein levels of dynamin 1 in the mouse hippocampal formation. In addition, the effects of dynasore, an inhibitor of dynamin 1, on the hippocampal dependent memory were determined to elucidate the correlation between dynamin 1 and memory. In the training phase of the Morris water maze task, the mean escape latency of the aged group (24 months old) was significantly longer, compared with that of the adult group (4 months old), although the average swimming speed and the total distance traveled during the probe trial were similar in the two groups. In the aged group, the time spent locating the target platform was significantly longer and the time spent in the correct quadrant was significantly shorter, compared with those in the adult group. In the adult group, a moderate level of dynamin 1 was detected in the hippocampal CA1 and CA3 regions, and in the dentate gyrus. In the aged group, the immunoreactivity of dynamin 1 was almost eliminated in the CA3 region and the dentate gyrus. In addition, the protein levels of dynamin 1 in the brain were significantly lower in the aged group, compared with those in the adult group. The direct infusion of dynasore, significantly reduced the contextual memory, compared with that of animals in the vehicle‑treated group. These results suggested that dynamin 1 was susceptible to the aging process, and that a reduction in dynamin 1 may result in hippocampal‑dependent memory deficits by disrupting endocytosis and the release of neurotransmitters.

  16. Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo

    Directory of Open Access Journals (Sweden)

    Richa Rikhy


    Full Text Available The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

  17. Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo (United States)

    Rikhy, Richa; Mavrakis, Manos; Lippincott-Schwartz, Jennifer


    ABSTRACT The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo. PMID:25661871

  18. Inhibition of the 20S proteosome by a protein proteinase inhibitor: evidence that a natural serine proteinase inhibitor can inhibit a threonine proteinase. (United States)

    Yabe, Kimihiko; Koide, Takehiko


    The 20S proteasome (20S) is an intracellular threonine proteinase (Mr 750,000) that plays important roles in many cellular regulations. Several synthetic peptide inhibitors and bacteria-derived inhibitors such as lactacystin and epoxomicin have been identified as potent proteasome inhibitors. However, essentially no protein proteinase inhibitor has been characterized. By examining several small size protein proteinase inhibitors, we found that a well-known serine proteinase inhibitor from bovine pancreas, basic pancreatic trypsin inhibitor (BPTI), inhibits the 20S in vitro and ex vivo. Inhibition of the 20S by BPTI was time- and concentration-dependent, and stoichiometric. To inhibit the 20S activity, BPTI needs to enter into the interior of the 20S molecule. The molar ratio of BPTI to the 20S in the complex was estimated as approximately six BPTI to one 20S, thereby two sets of three peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) of the 20S were all inhibited. These results indicate that an entrance hole to the 20S formed by seven alpha-subunits is sufficiently large for BPTI to enter. This report is essentially the initial description of the inhibition of a threonine proteinase by a protein serine proteinase inhibitor, suggesting a common mechanism of inhibition between serine and threonine proteinases by a natural protein proteinase inhibitor.

  19. Inhibition of microglial inflammation by the MLK inhibitor CEP-1347. (United States)

    Lund, Søren; Porzgen, Peter; Mortensen, Anne Louise; Hasseldam, Henrik; Bozyczko-Coyne, Donna; Morath, Siegfried; Hartung, Thomas; Bianchi, Marina; Ghezzi, Pietro; Bsibsi, Malika; Dijkstra, Sipke; Leist, Marcel


    CEP-1347 is a potent inhibitor of the mixed lineage kinases (MLKs), a distinct family of mitogen-activated protein kinase kinase kinases (MAPKKK). It blocks the activation of the c-Jun/JNK apoptotic pathway in neurons exposed to various stressors and attenuates neurodegeneration in animal models of Parkinson's disease (PD). Microglial activation may involve kinase pathways controlled by MLKs and might contribute to the pathology of neurodegenerative diseases. Therefore, the possibility that CEP-1347 modulates the microglial inflammatory response [tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1)] was explored. Indeed, the MLK inhibitor CEP-1347 reduced cytokine production in primary cultures of human and murine microglia, and in monocyte/macrophage-derived cell lines, stimulated with various endotoxins or the plaque forming peptide Abeta1-40. Moreover, CEP-1347 inhibited brain TNF production induced by intracerebroventricular injection of lipopolysaccharide in mice. As expected from a MLK inhibitor, CEP-1347 acted upstream of p38 and c-Jun activation in microglia by dampening the activity of both pathways. These data imply MLKs as important, yet unrecognized, modulators of microglial inflammation, and demonstrate a novel anti-inflammatory potential of CEP-1347.

  20. CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany); Poehlmann, Stefan [Institute of Virology, Hannover Medical School, 30625 Hannover (Germany); Holland, Gudrun; Bannert, Norbert [Robert Koch-Institute, Center for Biological Security 4, 13353 Berlin (Germany); Bogner, Elke [Institute of Virology, Charite University Hospital, 10117 Berlin (Germany); Schmidt, Barbara, E-mail: [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany)


    Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

  1. HIV-1 stimulates nuclear entry of amyloid beta via dynamin dependent EEA1 and TGF-β/Smad signaling

    Energy Technology Data Exchange (ETDEWEB)

    András, Ibolya E., E-mail:; Toborek, Michal, E-mail:


    Clinical evidence indicates increased amyloid deposition in HIV-1-infected brains, which contributes to neurocognitive dysfunction in infected patients. Here we show that HIV-1 exposure stimulates amyloid beta (Aβ) nuclear entry in human brain endothelial cells (HBMEC), the main component of the blood–brain barrier (BBB). Treatment with HIV-1 and/or Aβ resulted in concurrent increase in early endosomal antigen-1 (EEA1), Smad, and phosphorylated Smad (pSmad) in nuclear fraction of HBMEC. A series of inhibition and silencing studies indicated that Smad and EEA1 closely interact by influencing their own nuclear entry; the effect that was attenuated by dynasore, a blocker of GTP-ase activity of dynamin. Importantly, inhibition of dynamin, EEA1, or TGF-β/Smad effectively attenuated HIV-1-induced Aβ accumulation in the nuclei of HBMEC. The present study indicates that nuclear uptake of Aβ involves the dynamin-dependent EEA1 and TGF-β/Smad signaling pathways. These results identify potential novel targets to protect against HIV-1-associated dysregulation of amyloid processes at the BBB level. - Highlights: • HIV-1 induces nuclear accumulation of amyloid beta (Aβ) in brain endothelial cells. • EEA-1 and TGF-Β/Smad act in concert to regulate nuclear entry of Aβ. • Dynamin appropriates the EEA-1 and TGF-Β/Smad signaling. • Dynamin serves as a master regulator of HIV-1-induced nuclear accumulation of Aβ.

  2. Endocytic Trafficking of HIV gp120 is Mediated by Dynamin and Plays a Role in gp120 Neurotoxicity. (United States)

    Wenzel, Erin D; Bachis, Alessia; Avdoshina, Valeria; Taraballi, Francesca; Tasciotti, Ennio; Mocchetti, Italo


    Neurons that endocytose the human immunodeficiency virus-1 (HIV) protein gp120 exhibit neurite retraction and activation of caspase-3, suggesting that the endocytic process may be crucial for gp120-mediated neuronal injury. The goal of this study is to demonstrate that internalization and accumulation of gp120 play a role in its neurotoxic effects. In mammalian cells, endocytosis is primarily a dynamin-dependent process. To establish whether gp120 is endocytosed in a dynamin-dependent manner, we used fibroblasts in which deletion of dynamins was induced by tamoxifen. We observed a robust reduction of intracellular gp120 immunoreactivity in tamoxifen-treated cells. To examine whether endocytosis of gp120 is crucial for its neurotoxic effect, we blocked gp120 internalization into primary rat cortical neurons by dynasore, an inhibitor of the dynamin GTP-ase activity. We found that dynasore blocks both gp120 internalization and neurotoxicity. We then utilized gp120-loaded mesoporous silica nanoparticles to deliver gp120 intracellularly. We established that once internalized, gp120 is neurotoxic regardless of chemokine receptor activation. Our data suggest that dynamin-dependent endocytosis of gp120 is critical for its neurotoxicity.

  3. A Review of CO2 Corrosion Inhibition by Imidazoline-based Inhibitor

    Directory of Open Access Journals (Sweden)

    Jaal Rafida Ahmad


    Full Text Available Carbon dioxide (CO2 corrosion is one of the most significant forms of attack in the oil and gas production and transportation systems. Corrosion inhibitors have been widely used in an effort to reduce the detrimental effect of the corrosion process. Different types of corrosion inhibitors have been applied for this purpose. The most frequent is the imidazoline-based inhibitors (IM, owing to their good adsorption characteristics and film-forming capability. Albeit their extensive use, their inhibition mechanism is not fully understood. This paper highlights the inhibition mechanism of IM and also the factors that contribute to its inhibition mechanism.

  4. Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways. (United States)

    Huang, Haidong; Feng, Xiuyan; Zhuang, Jieqiu; Fröhlich, Otto; Klein, Janet D; Cai, Hui; Sands, Jeff M; Chen, Guangping


    Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH(2) and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH(2) and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.

  5. Evaluation and Structural Basis for the Inhibition of Tankyrases by PARP Inhibitors. (United States)

    Haikarainen, Teemu; Narwal, Mohit; Joensuu, Päivi; Lehtiö, Lari


    Tankyrases, an enzyme subfamily of human poly(ADP-ribosyl)polymerases, are potential drug targets especially against cancer. We have evaluated inhibition of tankyrases by known PARP inhibitors and report five cocrystal structures of the most potent compounds in complex with human tankyrase 2. The inhibitors include the small general PARP inhibitors Phenanthridinone, PJ-34, and TIQ-A as well as the more advanced inhibitors EB-47 and rucaparib. The compounds anchor to the nicotinamide subsite of tankyrase 2. Crystal structures reveal flexibility of the ligand binding site with implications for drug development against tankyrases and other ADP-ribosyltransferases. EB-47 mimics the substrate NAD(+) and extends from the nicotinamide to the adenosine subsite. The clinical ARTD1 inhibitor candidate rucaparib was the most potent tankyrase inhibitor identified (24 and 14 nM for tankyrases), which indicates that inhibition of tankyrases would affect the cellular responses of this compound.

  6. Dynamin2 organizes lamellipodial actin networks to orchestrate lamellar actomyosin.

    Directory of Open Access Journals (Sweden)

    Manisha Menon

    Full Text Available Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.

  7. Inhibition of tryptase release from human colon mast cells by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-Heng He; Hua Xie


    AIM: To investigate the ability of protease inhibitors to modulate tryptase release from human colon mast cells.METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors,and tryptase release was determined.RESULTS: IgE dependent tryptase release from colon mast cells was inhibited by up to approximately 37%, 40% and 36.6% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, the inhibitors of tryptase leupeptin, N-tosyl-L-lysine chloromethyl ketone (TLCK) and lactoferrin were also able to inhibit anti-IgE induced tryptase release by a maximum of 39.4%,47.6% and 36.6%, respectively. The inhibitory actions of chymase inhibitors, but not tryptase inhibitors on colon mast cells were enhanced by preincubation of them with cells for 20 min before challenged with anti-IgE. At a concentration of 10 μg/mL, protamine was able to inhibit anti-IgE and calcium ionophore induced tryptase release. However, at 100 μg/mL, protamine elevated tryptase levels in supernatants.A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced tryptase release. The significant inhibition of calcium ionophore induced tryptase release was also observed with the inhibitors of tryptase and chymase examined. The inhibitors tested by themselves did not stimulate tryptase release from colon mast cells.CONCLUSION: It was demonstrated for the first time that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced tryptase release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

  8. Acute inhibition of corticosteroidogenesis by inhibitors of calmodulin action. (United States)

    Carsia, R V; Moyle, W R; Wolff, D J; Malamed, S


    To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED50 of ACTH stimulation (3.2 X 10(3) pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 microM, a value close to the dissociation constant of the CP-calmodulin complex (5.3 microM). Concentrations greater than 40 microM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca2+ concentration, suggesting that the drug did not compete with Ca2+ directly. Concentrations of CP greater than 20 microM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 microM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine greater than CP greater than haloperidol greater than chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.

  9. Inhibiting the inhibitors: retro-inverso Smac peptides. (United States)

    Hossbach, Julia; Michalsky, Elke; Henklein, Peter; Jaeger, Marten; Daniel, Peter T; Preissner, Robert


    Resistance against apoptosis-inducing anti-cancer drugs remains a severe problem in therapy. One reason is the overexpression of inhibitors of apoptosis proteins (IAPs), a group of proteins responsible for the prevention of apoptosis induction by inactivation of initiator caspases. The natural inhibitor of the IAPs is the protein Smac, which impedes the binding to the caspases. Although Smac is a potent inhibitor, Smac peptides are not very stable in vivo and thus not applicable in therapy. Bioinformatical methods were applied to design Smac-derived peptides to break the therapy resistance in IAP high-expressing tumor cells. The exchange of amino acids in the Smac peptides AVPI and AVPF against unnatural amino acids leads to an improvement of the apoptosis sensitivity. The variety of Smac peptides was filtered by computational docking. Moreover, Smac-derived peptides with sufficient binding to the IAPs were tested in IAP-expressing Hodgkin Lymphoma cell lines.

  10. Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?


    Liu, Jiwen; Lee, TaeWeon; DeFronzo, Ralph A.


    Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokineti...

  11. What determines the inhibition effectiveness of ATA, BTAH, and BTAOH corrosion inhibitors on copper? (United States)

    Kokalj, Anton; Peljhan, Sebastijan; Finsgar, Matjaz; Milosev, Ingrid


    Three corrosion inhibitors for copper-3-amino-1,2,4-triazole (ATA), benzotriazole (BTAH), and 1-hydroxybenzotriazole (BTAOH)-were investigated by corrosion experiments and atomistic computer simulations. The trend of corrosion inhibition effectiveness of the three inhibitors on copper in near-neutral chloride solution is determined experimentally as BTAH ≳ ATA ≫ BTAOH. A careful analysis of the results of computer simulations based on density functional theory allowed to pinpoint the superior inhibiting action of BTAH and ATA as a result of their ability to form strong N-Cu chemical bonds in deprotonated form. While these bonds are not as strong as the Cl-Cu bonds, the presence of solvent favors the adsorption of inhibitor molecules onto the surface due to stronger solvation of the Cl(-) anions. Moreover, benzotriazole displays the largest affinity among the three inhibitors to form intermolecular aggregates, such as [BTA-Cu](n) polymeric complex. This is another factor contributing to the stability of the protective inhibitor film on the surface, thus making benzotriazole an outstanding corrosion inhibitor for copper. These findings cannot be anticipated on the basis of inhibitors' molecular electronic properties alone, thus emphasizing the importance of a rigorous modeling of the interactions between the components of the corrosion system in corrosion inhibition studies.

  12. The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Dormond-Meuwly, Anne; Roulin, Didier; Dufour, Marc; Benoit, Michael; Demartines, Nicolas [Department of Visceral Surgery, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Pavillon 3, Av. de Beaumont, 1011 Lausanne (Switzerland); Dormond, Olivier, E-mail: [Department of Visceral Surgery, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Pavillon 3, Av. de Beaumont, 1011 Lausanne (Switzerland)


    Highlights: {yields} Targeting mTOR in endothelial cell activates MAPK. {yields} Blocking MAPK enhances the anti-angiogenic effects of mTOR inhibitors. {yields} The anti-angiogenic efficacy of ATP-competitive inhibitors of mTOR is superior to that of rapamycin. -- Abstract: The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

  13. Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Han-qiu CHEN; Jian ZHENG


    AIM: To investigate the ability of proteinase inhibitors to modulate nucleated cell infiltration into the peritoneum of mice induced by tryptase and chymase. METHODS: Human lung tryptase and skin chymase were purified by a similar procedure involving high salt extraction, heparin agarose affinity chromatography followed by S-200 Sephacryl gel filtration chromatography. The actions of proteinase inhibitors on tryptase and chymase induced nucleated cell accumulation were examined with a mouse peritoneum model. RESULTS: A selective chymase inhibitor Z-Ile-GluPro-Phe-CO2Me (ZIGPPF) was able to inhibit approximately 90% neutrophil, 73% eosinophil, 87% lymphocyte and 60% macrophage accumulation induced by chymase at 16 h following injection. Soy bean trypsin inhibitor (SBTI), chymostatin, and α1-antitrypsin showed slightly less potency than ZIGPPF in inhibition of the actions of chymase. While all tryptase inhibitors tested were able to inhibit neutrophil, eosinophil, and macrophage accumulation provoked by tryptase at 16 h following injection, only leupeptin, APC366, and aprotinin were capable of inhibiting tryptase induced lymphocyte accumulation. The inhibitiors of tryptase tested were also able to inhibit tryptase induced neutrophil and eosinophil accumulation at 6 h following injection. When being injected alone, all inhibitors of chymase and tryptase at the concentrations tested by themselves had no significant effect on the accumulation of nucleated cells in the peritoneum of mice at both 6 h and 16 h. CONCLUSION: Proteinase inhibitors significantly inhibited tryptase and chymase-induced nucleated cell accumulation in vivo, and therefore they are likely to be developed as a novel class of anti-inflammatory drugs.

  14. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis. (United States)

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M


    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

  15. Oversulfated chondroitin sulfate inhibits the complement classical pathway by potentiating C1 inhibitor.

    Directory of Open Access Journals (Sweden)

    Zhao-Hua Zhou

    Full Text Available Oversulfated chondroitin sulfate (OSCS has become the subject of multidisciplinary investigation as a non-traditional contaminant in the heparin therapeutic preparations that were linked to severe adverse events. In this study, it was found that OSCS inhibited complement fixation on bacteria and bacterial lysis mediated by the complement classical pathway. The inhibition of complement by OSCS is not due to interference with antibody/antigen interaction or due to consumption of C3 associated with FXII-dependent contact system activation. However, OSCS complement inhibition is dependent on C1 inhibitor (C1inh since the depletion of C1inh from either normal or FXII-deficient complement plasma prevents OSCS inhibition of complement activity. Surface plasmon resonance measurements revealed that immobilized C1inhibitor bound greater than 5-fold more C1s in the presence of OSCS than in presence of heparin. Although heparin can also inhibit complement, OSCS and OSCS contaminated heparin are more potent inhibitors of complement. Furthermore, polysulfated glycosaminoglycan (PSGAG, an anti-inflammatory veterinary medicine with a similar structure to OSCS, also inhibited complement in the plasma of dogs and farm animals. This study provides a new insight that in addition to the FXII-dependent activation of contact system, oversulfated and polysulfated chondroitin-sulfate can inhibit complement activity by potentiating the classical complement pathway regulator C1inh. This effect on C1inh may play a role in inhibiting inflammation as well as impacting bacterial clearance.

  16. Inhibition of marine biofouling by bacterial quorum sensing inhibitors (United States)

    Dobretsov, Sergey; Teplitski, Max; Bayer, Mirko; Gunasekera, Sarath; Proksch, Peter; Paul, Valerie J


    Seventy eight natural products from chemical libraries containing compounds from marine organisms (sponges, algae, fungi, tunicates and cyanobacteria) and terrestrial plants, were screened for the inhibition of bacterial quorum sensing (QS) using a reporter strain Chromobacterium violaceum CV017. About half of the natural products did not show any QS inhibition. Twenty four percent of the tested compounds inhibited QS of the reporter without causing toxicity. The QS inhibitory activities of the most potent and abundant compounds were further investigated using the LuxR-based reporter E. coli pSB401 and the LasR-based reporter E. coli pSB1075. Midpacamide and tenuazonic acid were toxic to the tested reporters. QS-dependent luminescence of the LasR-based reporter, which is normally induced by N-3-oxo-dodecanoyl-L-homoserine lactone, was reduced by demethoxy encecalin and hymenialdisin at concentrations 46.6 μM and 15μM, respectively. Hymenialdisin, demethoxy encecalin, microcolins A and B and kojic acid inhibited responses of the LuxR-based reporter induced by N-3-oxo-hexanoyl-L-homoserine lactone at concentrations 40.2 μM, 2.2 μM, 1.5 μM, 15 μM and 36 μM, respectively. The ability to prevent microfouling by one of the compounds screened in this study (kojic acid; final concentrations 330 μM and 1 mM) was tested in a controlled mesocosm experiment. Kojic acid inhibited formation of microbial communities on glass slides, decreasing the densities of bacteria and diatoms in comparison with the control lacking kojic acid. The study suggests that natural products with QS inhibitory properties can be used for controlling biofouling communities. PMID:21882898

  17. Internalization of coxsackievirus A9 is mediated by {beta}2-microglobulin, dynamin, and Arf6 but not by caveolin-1 or clathrin. (United States)

    Heikkilä, Outi; Susi, Petri; Tevaluoto, Tuire; Härmä, Heidi; Marjomäki, Varpu; Hyypiä, Timo; Kiljunen, Saija


    Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin beta6 subunit inhibited virus proliferation, confirming that alphaVbeta6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of beta2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin alphaVbeta6, beta2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

  18. The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both? (United States)

    Nissan, Moriah H; Solit, David B


    Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

  19. Inhibition of arenavirus by A3, a pyrimidine biosynthesis inhibitor. (United States)

    Ortiz-Riaño, Emilio; Ngo, Nhi; Devito, Stefanie; Eggink, Dirk; Munger, Joshua; Shaw, Megan L; de la Torre, Juan Carlos; Martínez-Sobrido, Luis


    Arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The pyrimidine biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative- and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and pyrimidine biosynthesis inhibitors for the treatment of arenavirus infections.

  20. Expression of dynamin II in odontoblast during mouse tooth development. (United States)

    Oh, Jong-Hwa; Choi, Baik-Dong; Park, Jin-Ju; Jeong, Soon-Jeong; Kim, Jin-Soo; Kim, Jae-Duk; Lim, Do-Seon; Kim, Byung-Hoon; Cho, Yong-Ick; Jeong, Moon-Jin


    Odontoblasts secrete a collagen-based matrix and release numerous membrane-bound matrix vesicles, which are involved in dentin formation during tooth development. Dynamin II is a GTPase protein that contributes a variety of vesicular budding events, such as endocytotic membrane fission, caveolae internalization and protein trafficking in the Golgi apparatus. However, the expression and function of dynamin II in odontoblasts has not been reported. Therefore, this study examined the expression and possible role of dynamin II in odontoblasts during tooth development and mineralization. The levels of mRNA and protein expression in MDPC23 cells were significantly high at the early stages of differentiation and then decreased gradually thereafter. Immunohistochemistry showed that dynamin II was not expressed near the region of the odontoblasts at embryonic day 17 (E17) and E21. However, dynamin II was expressed strongly in the odontoblast layer at postnatal day 1 (PN1) and decreased gradually at PN3 and PN5. In addition, at PN15 in the functional stage, the dynamin II protein was also expressed in the odontoblast process as well as adjacent to the nuclear region. In conclusion, dynamin II may be involved in the transport of vesicles containing collageneous and non-collageneous proteins for dentin formation in odontoblast, suggesting that it is a good nanomolecule as a candidate to regulate the secretion of collagen on the bone and other nano material.

  1. Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

    Institute of Scientific and Technical Information of China (English)


    The irreversible modifying effects on Pst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou,C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB),diisopropyl fluorophosphate (DFP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3'-sulfonate (woodward's reagent K, WRK ) modify the lysine, cysine,serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity of Pst 1. Used with the irreversible inhibition theory,the apparent inhibition rate constant, A and the microcosmic inhibition rate constants, k+0 and k′ +o of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding.Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration of Pst I conformation and then influence the ability of Pst I recognizing and incising DNA specifically.

  2. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor. (United States)

    Wigand, R


    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  3. Inhibition of natural gas hydrates by means of kinetic inhibitors; Inhibierung von Erdgashydraten mit kinetischen Inhibitoren

    Energy Technology Data Exchange (ETDEWEB)

    Eberhardt, E.; Froemmel, J.; Hase, A. [Inst. fuer Erdoel- und Erdgasforschung, Clausthal-Zellerfeld (Germany)


    The use of kinetic inhibitors saves considerable costs as compared with thermodynamic inhibition. The effectivity of kinetic inhibitors can be examined by means of screening tests using an agitated autoclave. This contribution describes the experimental set-up and measuring methods used for this purpose and discusses the results obtained. (MSK) [Deutsch] Der Einsatz von kinetischen Inhibitoren fuehrt im Vergleich zur thermodynamischen Inhibition zu einer erheblichen Kostenreduzierung. Die Effektivitaet wird anhand von Screening-Versuchen in einem Ruehrautoklaven geprueft.Im Folgenden werden die Versuchsapparatur und die Messmethodik beschrieben. Ebenso werden die Ergebnisse diskutiert.

  4. Inhibition Performance of Enhanced-Mo Inhibitor for Carbon Steel in 55% LiBr Solution

    Institute of Scientific and Technical Information of China (English)

    LIANG Cheng-hao; HU Xian-qi


    The inhibition performance of enhanced-Mo inhibitor for carbon steel in 55% LiBr solution was measured by means of chemical immersion, electrochemical measurements, and physical detection technologies. Results indicated that enhanced-Mo inhibitor showed excellent inhibition performance of carbon steel in 55% LiBr solution, especially at high temperature. With increasing the temperature of solution from 160 ℃ to 240 ℃, the corrosion rates of carbon steel increased from 17.67 μm/a to 33.07 μm/a. Enhanced-Mo inhibitor might improve the anodic polarization performance of carbon steel and widen the passive potential region of carbon steel in 55% LiBr solution. Enhanced-Mo inhibitor belongs to anodic inhibitor. In 55% LiBr solution, the relationship between corrosion current density icorr and corrosion potential Ecorr of carbon steel accorded with the equation lgicorr=-2.66-3.54Ecorr, and the value of cathodic Tafel constant βc for the H2 reaction was 282 mVSCE. When 55% LiBr solution contained enhanced-Mo inhibitor, a passive film comprising Fe3O4 and MoO2 was formed on the carbon steel surface by electrochemical reactions. The corrosion of carbon steel might be retarded by this protective film, and the anticorrosion performance of carbon steel in 55% LiBr solution might be improved by enhanced-Mo inhibitor.

  5. Reduced release probability prevents vesicle depletion and transmission failure at dynamin mutant synapses. (United States)

    Lou, Xuelin; Fan, Fan; Messa, Mirko; Raimondi, Andrea; Wu, Yumei; Looger, Loren L; Ferguson, Shawn M; De Camilli, Pietro


    Endocytic recycling of synaptic vesicles after exocytosis is critical for nervous system function. At synapses of cultured neurons that lack the two "neuronal" dynamins, dynamin 1 and 3, smaller excitatory postsynaptic currents are observed due to an impairment of the fission reaction of endocytosis that results in an accumulation of arrested clathrin-coated pits and a greatly reduced synaptic vesicle number. Surprisingly, despite a smaller readily releasable vesicle pool and fewer docked vesicles, a strong facilitation, which correlated with lower vesicle release probability, was observed upon action potential stimulation at such synapses. Furthermore, although network activity in mutant cultures was lower, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity was unexpectedly increased, consistent with the previous report of an enhanced state of synapsin 1 phosphorylation at CaMKII-dependent sites in such neurons. These changes were partially reversed by overnight silencing of synaptic activity with tetrodotoxin, a treatment that allows progression of arrested endocytic pits to synaptic vesicles. Facilitation was also counteracted by CaMKII inhibition. These findings reveal a mechanism aimed at preventing synaptic transmission failure due to vesicle depletion when recycling vesicle traffic is backed up by a defect in dynamin-dependent endocytosis and provide new insight into the coupling between endocytosis and exocytosis.

  6. Single-membrane-bounded peroxisome division revealed by isolation of dynamin-based machinery. (United States)

    Imoto, Yuuta; Kuroiwa, Haruko; Yoshida, Yamato; Ohnuma, Mio; Fujiwara, Takayuki; Yoshida, Masaki; Nishida, Keiji; Yagisawa, Fumi; Hirooka, Shunsuke; Miyagishima, Shin-ya; Misumi, Osami; Kawano, Shigeyuki; Kuroiwa, Tsuneyoshi


    Peroxisomes (microbodies) are ubiquitous single-membrane-bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.

  7. Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments. (United States)

    Alam, N; Gourinath, S; Dey, S; Srinivasan, A; Singh, T P


    The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.

  8. Cyclooxygenase-2 inhibitor inhibits hippocampal synaptic reorganization in pilocarpine-induced status epilepticus rats

    Institute of Scientific and Technical Information of China (English)

    Hai-ju ZHANG; Ruo-peng SUN; Ge-fei LEI; Lu YANG; Chun-xi LIU


    Objective: To examine modulations caused by cyclooxygenase-2 (COX-2) inhibitors on altered microenvironments and overbalanced neurotransmitters in pilocarpine-induced epileptic status rats and to investigate possible mechanisms. Methods:Celecoxib (a COX-2 inhibitor) was administered 45 min prior to pilocarpine administration. The effects of COX-2 inhibitors on mIPSCs (miniature GABAergic inhibitory postsynaptic currents) of CA3 pyramidal cells in the hippocampus were recorded. Expressions of COX-2, c-Fos, newly generated neurons, and activated microgliosis wore analyzed by immunohistochemistry, and expressions of α-subunit of γ-amino butyric acid (GABAA) receptors and mitogen-activated protein kinase/extracellular sig-nal-regulated protein kinase (MAPK/ERK) activity were detected by Western blotting. Results: Pretreatment with celecoxib showed protection against pilocarpine-induced seizures. Celecoxib prevented microglia activation in the hilus and inhibited the abnormal neurogenesis and astrogliosis in the hippocampus by inhibiting MAPK/ERK activity and c-Fos transcription. Celecoxib also up-regulated the expression of GABAA receptors. NS-398 (N-2-cyclohexyloxy-4-nitrophenyl-methanesuifonamide), another COX-2 inhibitor, enhanced the frequency and decay time of mIPSCs. Conclusion: The COX-2 inhibitor celecoxib decreased neuronal excitability and prevented epileptogenesis in pilocarpine-induced status epilepticus rats. Celecoxib regulates synaptic reorganization by inhibiting astrogliosis and ectopic neurogenesis by attenuating MAPK/ERK signal activity, mediated by a GABAergic mechanism.

  9. Angiotensin-converting enzyme inhibition studies by natural leech inhibitors by capillary electrophoresis and competition assay. (United States)

    Deloffre, Laurence; Sautiere, Pierre-Eric; Huybrechts, Roger; Hens, Korneel; Vieau, Didier; Salzet, Michel


    A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.

  10. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. (United States)

    Bergman, Joel A; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M; Kozikowski, Alan P


    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

  11. Ruthenium-containing P450 inhibitors for dual enzyme inhibition and DNA damage. (United States)

    Zamora, Ana; Denning, Catherine A; Heidary, David K; Wachter, Erin; Nease, Leona A; Ruiz, José; Glazer, Edith C


    Cytochrome P450s are key players in drug metabolism, and overexpression in tumors is associated with significant resistance to many medicinal agents. Consequently, inhibition of P450s could serve as a strategy to restore drug efficacy. However, the widespread expression of P450s throughout the human body and the critical roles they play in various biosynthetic pathways motivates the development of P450 inhibitors capable of controlled local administration. Ruthenium complexes containing P450 inhibitors as ligands were synthesized in order to develop pro-drugs that can be triggered to release the inhibitors in a spatially and temporally controlled fashion. Upon light activation the compounds release ligands that directly bind and inhibit P450 enzymes, while the ruthenium center is able to directly damage DNA.

  12. Osteoblast differentiation and migration are regulated by Dynamin GTPase activity



    Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased...

  13. Lovastatin inhibits VEGFR and AKT activation: synergistic cytotoxicity in combination with VEGFR inhibitors.

    Directory of Open Access Journals (Sweden)

    Tong T Zhao

    Full Text Available BACKGROUND: In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR. Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR and EGFR share similar activation, internalization and downstream signaling characteristics. METHODOLOGY/PRINCIPAL FINDINGS: The VEGFRs, particularly VEGFR-2 (KDR, Flt-1, play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM, also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. CONCLUSIONS/SIGNIFICANCE: These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors.

  14. New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans. (United States)

    Zhang, Qiong; Nguyen, Thao; McMichael, Megan; Velu, Sadanandan E; Zou, Jing; Zhou, Xuedong; Wu, Hui


    Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.

  15. General amyloid inhibitors? A critical examination of the inhibition of IAPP amyloid formation by inositol stereoisomers.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Islet amyloid polypeptide (IAPP or amylin forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.

  16. Specific inhibition of caspase-3 by a competitive DARPin: molecular mimicry between native and designed inhibitors. (United States)

    Schroeder, Thilo; Barandun, Jonas; Flütsch, Andreas; Briand, Christophe; Mittl, Peer R E; Grütter, Markus G


    Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways.

  17. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors. (United States)

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N


    RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.

  18. Momordica charantia trypsin inhibitorinhibits growth and development of Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    Manasi Alok Telang; Prashant Pyati; Mohini Sainani; Vidya Shrikant Gupta; Ashok Prabhakar Giri


    Bitter gourd (Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs),which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera.In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-Ⅱ,its cloning and expression as a recombinant protein using Pichia pastoris have been reported.Recombinant McTI-Ⅱinhibited bovine trypsin at 1:1 molar ratio,as expected,but did not inhibit chymotrypsin or elastase.McTI-Ⅱalso strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H.armigera larvae.The insect larvae fed with McTI-Ⅱ-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding.Moreover,ingestion of McTI-Ⅱresulted in 23% mortality in the larval population.The strong antimetabolic activity of McTI-Ⅱtoward H.armigera indicates its probable use in developing insect tolerance in susceptible plants.

  19. New method for the determination of the half inhibition concentration (IC50) of cholinesterase inhibitors. (United States)

    Kovárová, Markéta; Komers, Karel; Stepánková, Sárka; Parík, Patrik; Cegan, Alexander


    A new and simple analytical method is described for the determination of the IC50 values of the inhibitors of the hydrolysis of acetylcholine (ACh) or acetylthiocholine (ATCh) by cholinesterases. The method is based on monitoring the time course of the pH value during the uninhibited and inhibited reaction. It requires only a pH meter with a suitable pH measuring cell and a small thermostated stirred batch reactor. The method has been validated for twelve different types of cholinesterase inhibitors. The determined IC50 values are comparable to those obtained by independent, more complicated, and expensive methods (Ellman's and pH-stat).

  20. Inhibition of histamine release from human mast cells by natural chymase inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Hua XIE; Xiao-jun ZHANG; Xian-jie WANG


    AIM: To investigate the ability of natural chymase inhibitors to modulate histamine release from human mast cells.METHODS: Enzymatically dispersed cells from human lung, tonsil, and skin were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of the natural chymase inhibitors secretory leukocyte protease inhibitor (SLPI) and α1-antitrypsin, then histamine release was determined. RESULTS: IgE-dependent histamine release from lung, tonsil, and skin mast cells were inhibited by up to 70 %, 61%, and 62%, respectively following incubation with α1-antitrypsin (5000 nmol/L). SLPI 5000 nmol/L was also able to inhibit anti-IgEdependent histamine released from lung, tonsil and skin mast cells by up to approximately 72%, 67%, and 58%,respectively. While neither α1-antitrypsin nor SLPI by themselves altered histamine release from lung, tonsil and skin mast cells, they were able to inhibit calcium ionophore-induced histamine release from lung and tonsil mast cells. CONCLUSION: Both α1-antitrypsin and SLPI could potently inhibit IgE-dependent and calcium ionophoreinduced histamine release from dispersed human lung, tonsil, and skin mast cells in a concentration-dependent manner, which suggested that they were likely to play a protective role in mast cell associated diseases including allergy.

  1. Inhibition of phosphatidylinositol-specific phospholipase C: studies on synthetic substrates, inhibitors and a synthetic enzyme. (United States)

    Vizitiu, D; Kriste, A G; Campbell, A S; Thatcher, G R


    Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.

  2. Dynamics of dynamin during clathrin mediated endocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Joshua Z Rappoport

    Full Text Available BACKGROUND: Members of the dynamin super-family of GTPases are involved in disparate cellular pathways. Dynamin1 and dynamin2 have been implicated in clathrin-mediated endocytosis. While some models suggest that dynamin functions specifically at the point of vesicle fission, evidence also exists for a role prior to fission during vesicle formation and it is unknown if there is a role for dynamin after vesicle fission. Although dynamin2 is ubiquitously expressed, dynamin1 is restricted to the nervous system. These two structurally similar endocytic accessory proteins have not been studied in cells that endogenously express both. METHODOLOGY/PRINCIPAL FINDINGS: The present study quantitatively assesses the dynamics of dynamin1 and dynamin2 during clathrin-mediated endocytosis in PC12 cells, which endogenously express both proteins. Both dynamin isoforms co-localized with clathrin and showed sharp increases in fluorescence intensity immediately prior to internalization of the nascent clathrin-coated vesicle. The fluorescence intensity of both proteins then decreased with two time constants. The slower time constant closely matched the time constant for the decrease of clathrin intensity and likely represents vesicle movement away from the membrane. The faster rate may reflect release of dynamin at the neck of nascent vesicle following GTP hydrolysis. CONCLUSIONS/SIGNIFICANCE: This study analyses the role of dynamin in clathrin-mediated endocytosis in a model for cellular neuroscience and these results may provide direct evidence for the existence of two populations of dynamin associated with nascent clathrin-coated vesicles.

  3. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis (United States)

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire


    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  4. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma. (United States)

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S


    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy.

  5. Inhibitors of 5-lipoxygenase inhibit expression of intercellular adhesion molecule-1 in human melanoma cells

    Institute of Scientific and Technical Information of China (English)

    Yin WANG; Bin ZHOU; Ji LI; Yong-bing CAO; Xin-sheng CHEN; Ming-he CHENG; Ming YIN


    AIM: To study the effect of 5-lipoxygenase inhibitors on the expression of intercellular adhesion molecule-1 (ICAM-1) in melanoma cells. METHODS: ICAM-1 protein of human melanoma cell a375 was detected by enzyme-linked immunosorbent, flow cytometry and Western blot analysis. Level of ICAM-1 mRNA in a375 was evaluated by Northern blot analysis. Adhesion of a375 to endothelial cell EC304 was analyzed by isotopic tracing. RESULTS:5-Lipoxygenase inhibitors nordihydroguaiaretic acid, AA861 and MK886, could suppress the expression of ICAM-1 protein as well as of its mRNA in a375 cells and reduce the adhesion of a375 to EC304. CONCLUSION:5-Lipoxygenase inhibitors can inhibit the expression of ICAM-1 in human melanoma cells and may be valuable for treatment of melanoma metastasis.

  6. Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

    Directory of Open Access Journals (Sweden)

    Jian Hua Qi

    Full Text Available Tissue inhibitors of metalloproteinases (TIMPs while originally characterized as inhibitors of matrix metalloproteinases (MMPs have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3 is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

  7. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)


    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  8. A combination strategy to inhibit Pim-1: synergism between noncompetitive and ATP-competitive inhibitors. (United States)

    Mori, Mattia; Tintori, Cristina; Christopher, Robert Selwyne Arul; Radi, Marco; Schenone, Silvia; Musumeci, Francesca; Brullo, Chiara; Sanità, Patrizia; Delle Monache, Simona; Angelucci, Adriano; Kissova, Miroslava; Crespan, Emmanuele; Maga, Giovanni; Botta, Maurizio


    Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.

  9. Study on Surface Adsorption and Inhibition Behavior of Corrosion Inhibitors Contained in Copper Foil Rolling Oil

    Institute of Scientific and Technical Information of China (English)

    Xiong Sang; Sun Jianlin; Jiang Wei; Xu Yang; Zeng Yingfeng; Xia Lei


    Adsorption and inhibition behavior of 2,5-bis(ethyldisulfanyl)-1,3,4-thiadiazole (DMTDA) andN-((6-methyl-1H -benzo[d][1,2,3]triazol-1-yl)methyl)-N-octyloctan-1-amine (EAMBA) as corrosion inhibitors contained in copper foil roll-ing oil have been investigated using gravimetric and electrochemical techniques. Meanwhile, scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) have been employed to observe the surface topography and analyze the components on copper foil. The results show that the rolling oil containing DMTDA and EAMBA can signiifcantly decrease the dissolution rate and increase the inhibition efifciency of samples, especially in the case of best compounded rolling oil system. The SEM and EDS investigations also conifrmed that the protection of the copper foil surface is achieved by strong adsorption of the molecules which can prevent copper from being corroded easily. Reactivity descriptors of the corrosion inhibitors have been calculated by the density functional theory (DFT) and the reactivity has been analyzed through the molecular orbital and Fukui indices. Active sites of inhibitor are mainly concentrated on the ring and the polar functional groups, and in the meanwhile, the distribution is helpful to form coordination and backbonding among molecules and then to form stable adsorption on the metal surface. And this work provides theoretical evidence for the selection of corrosion inhibitors contained in copper foil rolling oil.

  10. Multifaceted mechanisms of HIV inhibition and resistance to CCR5 inhibitors PSC-RANTES and Maraviroc. (United States)

    Lobritz, Michael A; Ratcliff, Annette N; Marozsan, Andre J; Dudley, Dawn M; Tilton, John C; Arts, Eric J


    Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

  11. Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells. (United States)

    Ghoshal, Pushpankur; Rajendran, Mythilypriya; Odo, Nadine; Ikuta, Tohru


    Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.

  12. Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs.

    Directory of Open Access Journals (Sweden)

    Anne T Nies

    Full Text Available Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3, which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50 were in the low micromolar range (3-36 µM and thereby in the range of IC(50 values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.

  13. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail:


    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

  14. Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions

    DEFF Research Database (Denmark)

    Boissy, P; Levin Andersen, Thomas; Lund, T


    Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib...... on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3h pulse with 25nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone...... resorption, TRAcP release, and RANKL-induced NF-kappaB translocation into nuclei, an event dependent on proteasomes and critical for osteoclast function. The effect on TRAcP was maximal during the first 24h post-pulse, and then tended to subside. Importantly, applying this pulse treatment to cultured myeloma...

  15. Regulation of dynamin family proteins by post-translational modifications

    Indian Academy of Sciences (India)



    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agentsinvolved in several biological processes such as endocytosis, maintenance of organelle morphology and viralresistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion ortubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulatedby various post-translational modifications that affect their GTPase activity, multimerization or membrane association.Recently, several reports have demonstrated variety of such modifications providing a better understanding of themechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In thisreview, we discuss major post-translational modifications along with their roles in the mechanism of dynaminfunctions and implications in various cellular processes.

  16. Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo. (United States)

    Votta, B J; Levy, M A; Badger, A; Bradbeer, J; Dodds, R A; James, I E; Thompson, S; Bossard, M J; Carr, T; Connor, J R; Tomaszek, T A; Szewczuk, L; Drake, F H; Veber, D F; Gowen, M


    We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

  17. Comparison of iNOS inhibition by antisense and pharmacological inhibitors after spinal cord injury. (United States)

    Pearse, D D; Chatzipanteli, K; Marcillo, A E; Bunge, M B; Dietrich, W D


    Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation during pathological conditions. We examined, through the use of selective iNOS inhibitors, the role of iNOS in specific pathophysiological processes after spinal cord injury (SCI), including astrogliosis, blood-spinal cord barrier (BSCB) permeability, polymorphonuclear leukocyte infiltration, and neuronal cell death. Administration of iNOS antisense oligonucleotides (ASOs) (intraspinally at 3 h) or the pharmacological inhibitors, N-[3(Aminomethyl) benzyl] acetamidine (1400 W) (i.v./i.p. 3 and 9 h) or aminoguanidine (i.p. at 3 and 9 h) after moderate contusive injury decreased the number of iNOS immunoreactive cells at the injury site by 65.6% (iNOS ASOs), 62.1% (1400 W), or 59% (aminoguanidine) 24 h postinjury. iNOS activity was reduced 81.8% (iNOS ASOs), 56.7% (1400 W), or 67.9% (aminoguanidine) at this time. All iNOS inhibitors reduced the degree of BSCB disruption (plasma leakage of rat immunoglobulins), with iNOS ASO inhibition being more effective (reduced by 58%). Neutrophil accumulation within the injury site was significantly reduced by iNOS ASOs and 1400 W by 78.8% and 20.9%, respectively. Increased astrogliosis was diminished with iNOS ASOs but enhanced following aminoguanidine. Detection of necrotic and apoptotic neuronal cell death by propidium iodide and an FITC-conjugated Annexin V antibody showed that iNOS inhibition could significantly retard neuronal cell death rostral and caudal to the injury site. These novel findings indicate that acute inhibition of iNOS is beneficial in reducing several pathophysiological processes after SCI. Furthermore, we demonstrate that the antisense inhibition of iNOS is more efficacious than currently available pharmacological agents.

  18. Zebrafish as a Model to Investigate Dynamin 2-Related Diseases


    Cinzia Bragato; Germano Gaudenzi; Flavia Blasevich; Giulio Pavesi; Lorenzo Maggi; Michele Giunta; Franco Cotelli; Marina Mora


    Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down...

  19. Dynamin 2 mutations cause sporadic centronuclear myopathy with neonatal onset. (United States)

    Bitoun, Marc; Bevilacqua, Jorge A; Prudhon, Bernard; Maugenre, Svetlana; Taratuto, Ana Lia; Monges, Soledad; Lubieniecki, Fabiana; Cances, Claude; Uro-Coste, Emmanuelle; Mayer, Michèle; Fardeau, Michel; Romero, Norma B; Guicheney, Pascale


    We report four heterozygous dynamin 2 (DNM2) mutations in five centronuclear myopathy patients aged 1 to 15 years. They all presented with neonatal hypotonia with weak suckling. Thereafter, their phenotype progressively improved. All patients demonstrated muscle weakness prominent in the lower limbs, and most of them also presented with facial weakness, open mouth, arched palate, ptosis, and ophthalmoparesis. Electrophysiology showed only myopathic changes, and muscle biopsies showed central nuclei and type 1 fiber hypotrophy and predominance. Our results expand the phenotypic spectrum of dynamin 2-related centronuclear myopathy from the classic mild form to the more severe neonatal phenotype.

  20. Cyclo-oxygenase 2 inhibitor, nabumetone, inhibits proliferation in chronic myeloid leukemia cell lines. (United States)

    Vural, Filiz; Ozcan, Mehmet Ali; Ozsan, Güner Hayri; Ateş, Halil; Demirkan, Fatih; Pişkin, Ozden; Undar, Bülent


    The anti-tumor effect of cyclo-oxygenase (COX) inhibitors has been documented in several studies. COX2 inhibitors have attracted more attention because of the fewer side-effects and the more prominent anti-tumor effects. However, experience with these drugs in hematological malignancies is limited. In our study, a potent COX2 inhibitor, nabumetone (NBT), was investigated for its anti-proliferative and apoptotic effects in K-562 and Meg-01 chronic myeloid leukemia blastic cell lines as a single agent or in combination with adriamycin (ADR) and interferon alpha (IFN-a). In these cell lines, a dose-dependent inhibition of proliferation was observed with NBT. We observed no significant apoptotic effect of NBT. However, NBT potentiated the apoptotic effect of ADR in the K-562 cell line. Bcl-2 expression was reduced by NBT (11% vs. 2%). The combination of NBT with IFN did not have any significant effect on the K-562 cell line. We suggest that NBT inhibits proliferation and potentiates the apoptotic effect of ADR in chronic myeloid leukemia cell lines.

  1. Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Graham, Mark E; Robinson, Phillip J


    , or peptides altered in hydrophilicity such as phosphopeptides. We used microcolumns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analyzed. MS revealed that some phosphopeptides...... did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve...... and doubly phosphorylated peptide in dynamin III, analogous to the dynamin I sequence. A pair of dynamin III phosphorylation sites were found at Ser-759 and Ser-763 by tandem MS. The results directly define the in vivo phosphorylation sites in dynamins I and III for the first time. The findings indicate...

  2. Investigation of scale inhibition mechanisms based on the effect of scale inhibitor on calcium carbonate crystal forms

    Institute of Scientific and Technical Information of China (English)

    ZHANG GuiCai; GE JiJiang; SUN MingQin; PAN BinLin; MAO Tao; SONG ZhaoZheng


    To probe the scale inhibition mechanisms, calcium carbonate scale occurring before and after the addition of scale inhibitors was collected. The results from scale SEM confirm that, without scale inhibitor, calcium carbonate scale shows rhombohedron and hexagon, which are the characteristic feathers of calcite. After addition of inhibitors, morphology of scale is changed, and the more efficient the scale inhibitor is, the more greatly the morphology is modified. To elucidate the scale constitute, they were further analyzed by FT-IR, XRD. Besides calcite, vaterite and aragonite occur in calcium carbonate scale after addition of inhibitors, and the higher scale inhibition efficiency is, the more vaterite presents in scale. It can be concluded that the alteration of morphology is ascribed to the change of crystal form. There are three stages in the crystallizing process including occurrence and disappearing of unstable phase, occurrence and disappearing of metastable phase, development of stable phase. Without scale inhibitors, metastable phases usually transform into stable phase, thus the main constitute of formed scale is calcite. When scale inhibitors are added, both formation and transformation of metastable phases are inhibited, which results in the occurrence of aragonite and vaterite. From the fact that more vaterite presents in scale with a more efficient scale inhibitor added, we can see that the function of scale inhibitor is realized mainly by controlling the crystallizing process at the second stage.

  3. Investigation of scale inhibition mechanisms based on the effect of scale inhibitor on calcium carbonate crystal forms

    Institute of Scientific and Technical Information of China (English)


    To probe the scale inhibition mechanisms,calcium carbonate scale occurring before and after the ad- dition of scale inhibitors was collected.The results from scale SEM confirm that,without scale inhibitor, calcium carbonate scale shows rhombohedron and hexagon,which are the characteristic feathers of calcite.After addition of inhibitors,morphology of scale is changed,and the more efficient the scale inhibitor is,the more greatly the morphology is modified.To elucidate the scale constitute,they were further analyzed by FT-IR,XRD.Besides calcite,vaterite and aragonite occur in calcium carbonate scale after addition of inhibitors,and the higher scale inhibition efficiency is,the more vaterite presents in scale.It can be concluded that the alteration of morphology is ascribed to the change of crystal form. There are three stages in the crystallizing process including occurrence and disappearing of unstable phase,occurrence and disappearing of metastable phase,development of stable phase.Without scale inhibitors,metastable phases usually transform into stable phase,thus the main constitute of formed scale is calcite.When scale inhibitors are added,both formation and transformation of metastable phases are inhibited,which results in the occurrence of aragonite and vaterite.From the fact that more vaterite presents in scale with a more efficient scale inhibitor added,we can see that the function of scale inhibitor is realized mainly by controlling the crystallizing process at the second stage.

  4. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II. (United States)

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma


    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

  5. Hybrid triazoles: Design and synthesis as potential dual inhibitor of growth and efflux inhibition in tuberculosis. (United States)

    Dixit, Prasad P; Dixit, Prashant P; Thore, Shivajirao N


    Efflux inhibition is proven bacterial machinery responsible for removal of bacterial wastage including antibiotics. Recently, efflux inhibitors (EI) have been tested with encouraging results as an adjuvant therapy for treatment of tuberculosis (TB). Although, EI have emerged as innovative approach of treatment for multi drug resistant (MDR) & extensively drug resistant tuberculosis (XDR-TB), toxicity profile limits their wider use. To address this issue, we have attempted synthesizing hybrid molecules those results by combining known EI and triazole. This synthesis was aimed to arrive at structure that possesses pharmacophore from known EI. Synthesized molecules were evaluated as growth inhibitors (GI) and Efflux inhibitor of TB initially against Mycobacterium smegmatis mc(2)155. Pharmacologically active compounds were then tested for their cytotoxicity to further narrow down search. Most active compounds 144, 145, 154 and 163 were then tested for their GEI action against Mycobacterium tuberculosis (Mtb). Synthesized compounds were also tested for their synergistic action with first line and second line anti-TB drugs and ethidium bromide (EtBr). We arrived at compound 135 as most potent dual inhibitor of tuberculosis.

  6. Is the analysis of molecular electronic structure of corrosion inhibitors sufficient to predict the trend of their inhibition performance

    Energy Technology Data Exchange (ETDEWEB)

    Kokalj, Anton, E-mail: tone.kokalj@ijs.s [Department of Physical and Organic Chemistry, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia)


    The often used approach in the corrosion inhibition studies employing quantum chemical calculations that relies on the correlation between molecular electronic structure parameters and inhibition effectiveness is critically examined. It is shown that the inhibition performance of three selected triazole-based corrosion inhibitors for copper - 3-amino-1,2,4-triazole (ATA), benzotriazole (BTAH), and 1-hydroxybenzotriazole (BTAOH) - cannot be explained on this basis in a sound manner. As the effectiveness of inhibitors is due to several phenomena, the outcome depends on the interplay between them and although molecular electronic parameters may provide many necessary elements, the involved effects can be estimated only approximately which may not always suffice. This supports the proposition that in general molecular electronic properties cannot be directly related to inhibition effectiveness - the actual relation is more involved - thus emphasizing the importance of a rigorous modeling of the inhibitor-surface interaction in the corrosion inhibition studies.

  7. Momordin Ic, a new natural SENP1 inhibitor, inhibits prostate cancer cell proliferation. (United States)

    Wu, Jingjing; Lei, Hu; Zhang, Jinfu; Chen, Xiangyun; Tang, Caixia; Wang, Weiwei; Xu, Hanzhang; Xiao, Weilie; Gu, Wenli; Wu, Yingli


    SUMO-specific protease 1 (SENP1), a member of the de-SUMOylation protease family, is elevated in prostate cancer (PCa) cells and is involved in PCa pathogenesis. Momordin Ιc (Mc), a natural pentacyclic triterpenoid, inhibited SENP1 in vitro, as reflected by reduced SENP1C-induced cleavage of SUMO2-ΔRanGAP1. Mc also altered the thermal stability of SENP1 in a newly developed cellular thermal shift assay, indicating that Mc directly interacts with SENP1 in PCa cells. Consistent with SENP1 inhibition, Mc increased SUMOylated protein levels, which was further confirmed by the accumulation of two known SUMOylated proteins, hypoxia inducible factor-1a and nucleus accumbens associated protein 1 in PC3 cells. Compared to LNCaP and normal prostate epithelial RWPE-1 cells, PC3 cells had higher levels of SENP1 mRNA and were more sensitive to Mc-induced growth inhibition. Mc also reduced SENP1 mRNA levels in PCa cells. Overexpression of SENP1 rescued PC3 cells from Mc-induced apoptosis. Finally, Mc suppressed cell proliferation and induced cell death in vivo in a xenograft PC3 tumor mouse model. These findings demonstrate that Mc is a novel SENP1 inhibitor with potential therapeutic value for PCa. Investigation of other pentacyclic triterpenoids may aid in the development of novel SENP1 inhibitor drugs.

  8. Inhibition mechanism exploration of investigational drug TAK-441 as inhibitor against Vismodegib-resistant Smoothened mutant. (United States)

    Ishii, Tsuyoshi; Shimizu, Yuji; Nakashima, Kosuke; Kondo, Shigeru; Ogawa, Kazumasa; Sasaki, Satoshi; Matsui, Hideki


    Hedgehog signaling is a driving force in medulloblastoma and basal cell carcinoma (BCC), making it an attractive therapeutic target. Vismodegib recently received FDA approval for the treatment of inoperable BCC, but a drug-resistant Smoothened (Smo) mutant (D473H) was identified in a clinical study. TAK-441 is a pyrrolo[3,2-c]pyridine-4-one derivative that potently inhibits Hh signal transduction and is currently under investigation in clinical trials. We demonstrated that TAK-441 inhibits reporter activity in D473H-transfected cells with an IC50 of 79nM, while Vismodegib showed an IC50=7100nM. In order to investigate the mode of inhibition, we evaluated the Smo inhibitors with three different binding assays, such as [(3)H]-TAK-441 membrane binding assay, affinity selection-MS detection assay, and bodipy-cylopamine whole cell assay. In three different assays, Vismodegib and cyclopamine showed lower affinity for the D473H mutant in comparison with wild-type Smo. On the other hand, TAK-441 showed almost equal binding affinity for the D473H mutant compared with wild-type Smo in the binding assays, although TAK-441 binds to the same binding site as two other well-known inhibitors. These in vitro findings suggest that TAK-441 has the potential for clinical use in cancers that are dependent on Hedgehog signaling, including wild-type tumors and Vismodegib-resistant D473H mutants.

  9. Structural Basis for Reversible and Irreversible Inhibition of Human Cathepsin L by their Respective dipeptidyl glyoxal and diazomethylketone Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    R Shenoy; J Sivaraman


    Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors - inhibitor 1, an {alpha}-keto-{beta}-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a Ki of 0.6 nM, is the most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 {angstrom} resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at {micro}M concentrations, was refined up to 1.76 {angstrom} resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 {beta}-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S' subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.

  10. Investigating isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins for ovarian cancer treatment

    Directory of Open Access Journals (Sweden)

    Chen C


    Full Text Available Chen Chen,1,* Jie Wu,2,* Pengfei Zhu,3 Congjian Xu,1 Liangqing Yao1 1Obstetrics and Gynecology Hospital and Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, 2Department of Chemistry, Fudan University, Shanghai, 3Department of Obstetrics and Gynecology, Shangyu City Hospital, Shangyu, Zhejiang Province, People’s Republic of China *These authors contributed equally to this work Objective: To discover novel isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins (IAP for the treatment of ovarian cancer. Methods: We first synthesized 533 isoquinoline derivatives, and screened them using CCK-8 to measure their antiproliferative activity. These compounds were further tested by Hoechst staining and flow cytometric analysis to assess proapoptotic activity. The in vivo antitumor efficacy and safety of the screened compounds were evaluated on the xenograft mouse model. Ki-67 staining and TUNEL assay were used to evaluate proliferation and apoptosis in the resected tumors, respectively. Western blot and polymerase chain reaction (PCR were conducted to evaluate the levels of proliferating cell nuclear antigen (PCNA, caspase-3, PARP, and IAP in resected tumors. Results: Compound B01002 and C26001 displayed antiproliferative and proapoptotic activity on SKOV3 ovarian cancer with an IC50 of 7.65 and 11.68 µg/mL, respectively. Both compounds inhibited tumor growth in a xenografted mouse model with good safety profiles, and tumor growth inhibition (TGI of B01002 and C26001 was 99.53% and 84.23%, respectively. Resected tumors showed that both compounds inhibited tumor cell proliferation and induced apoptosis in vivo. Caspase-3 and PARP were activated, whereas IAP proteins were downregulated at the protein level. Conclusion: Compound B01002 and C26001 could inhibit ovarian tumor growth and promote tumor apoptosis, partly by downregulating the IAPs, and, thus, might be promising candidates for treatment of ovarian

  11. Galectin-3 Inhibition by a Small-Molecule Inhibitor Reduces Both Pathological Corneal Neovascularization and Fibrosis (United States)

    Chen*, Wei-Sheng; Cao, Zhiyi; Leffler, Hakon; Nilsson, Ulf J.; Panjwani, Noorjahan


    Purpose Corneal neovascularization and scarring commonly lead to significant vision loss. This study was designed to determine whether a small-molecule inhibitor of galectin-3 can inhibit both corneal angiogenesis and fibrosis in experimental mouse models. Methods Animal models of silver nitrate cautery and alkaline burn were used to induce mouse corneal angiogenesis and fibrosis, respectively. Corneas were treated with the galectin-3 inhibitor, 33DFTG, or vehicle alone and were processed for whole-mount immunofluorescence staining and Western blot analysis to quantify the density of blood vessels and markers of fibrosis. In addition, human umbilical vein endothelial cells (HUVECs) and primary human corneal fibroblasts were used to analyze the role of galectin-3 in the process of angiogenesis and fibrosis in vitro. Results Robust angiogenesis was observed in silver nitrate–cauterized corneas on day 5 post injury, and markedly increased corneal opacification was demonstrated in alkaline burn–injured corneas on days 7 and 14 post injury. Treatment with the inhibitor substantially reduced corneal angiogenesis and opacification with a concomitant decrease in α-smooth muscle actin (α-SMA) expression and distribution. In vitro studies revealed that 33DFTG inhibited VEGF-A–induced HUVEC migration and sprouting without cytotoxic effects. The addition of exogenous galectin-3 to corneal fibroblasts in culture induced the expression of fibrosis-related proteins, including α-SMA and connective tissue growth factor. Conclusions Our data provide proof of concept that targeting galectin-3 by the novel, small-molecule inhibitor, 33DFTG, ameliorates pathological corneal angiogenesis as well as fibrosis. These findings suggest a potential new therapeutic strategy for treating ocular disorders related to pathological angiogenesis and fibrosis. PMID:28055102

  12. Dynamin GTPase Regulation is Altered by PH Domain Mutations Found in Centronuclear Myopathy Patients

    Energy Technology Data Exchange (ETDEWEB)

    Kenniston, J.; Lemmon, M


    The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal {alpha}-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

  13. Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible. (United States)

    Zhuo, Y; Gauthier, J-Y; Black, W C; Percival, M D; Duong, L T


    The cathepsin K (CatK) inhibitor odanacatib (ODN) is currently being developed for the treatment of osteoporosis. In clinical trials, efficacy and resolution of effect of ODN treatment on bone turnover biomarkers and accrued bone mass have been demonstrated. Here, we examine the effects of continuing treatment and discontinuation of ODN versus alendronate (ALN) on osteoclast (OC) function. First, accessibility and reversible engagement of active CatK in intracellular vesicles and resorption lacunae of actively resorbing OCs were demonstrated by the selective and reversible CatK inhibitors, BODIPY-L-226 (IC50=39nM) and L-873,724 (IC50=0.5nM). Next, mature human OCs on bone slices were treated with vehicle, ODN, or ALN for 2days, followed by either continuing with the same treatment, or replacement of the inhibitors by vehicle for additional times as specified per experimental conditions. Maintaining OCs on ODN or ALN significantly reduced CTx-I release compared to vehicle controls. However, only the treatment of OCs with ODN resulted in the formation of small shallow discrete resorption pits, retention of intracellular vesicles enriched with CatK and other lysosomal enzymes, increase in 1-CTP release and number of TRAP(+) OCs. Upon discontinuation of ODN treatment, OCs rapidly resumed bone resorption activity, as demonstrated by a return of OC functional markers (CTx-I, 1-CTP), cell number and size, morphology and number of resorption pits, and vesicular secretion of CatK toward the respective vehicle levels. As expected, discontinuation of ALN did not reverse the treatment-related inhibition of OC activity in the time frame of the experiment. In summary, this study demonstrated rapid kinetics of inhibition and reversibility of the effects of ODN on OC bone resorption, that differentiated the cellular mechanism of CatK inhibition from that of the bisphosphate antiresorptive ALN.

  14. Corrosion inhibition by inorganic cationic inhibitors on the high strength alumunium alloy, 2024-T3 (United States)

    Chilukuri, Anusha

    The toxicity and carcinogenic nature of chromates has led to the investigation of environmentally friendly compounds that offer good corrosion resistance to AA 2024-T3. Among the candidate inhibitors are rare earth metal cationic (REM) and zinc compounds, which have received much of attention over the past two decades. A comparative study on the corrosion inhibition caused by rare earth metal cations, Ce3+, Pr3+, La3+ and Zn2+ cations on the alloy was done. Cathodic polarization showed that these inhibitor ions suppress the oxygen reduction reaction (ORR) to varying extents with Zn2+ providing the best inhibition. Pr3+ exhibited windows of concentration (100-300 ppm) in which the corrosion rate is minimum; similar to the Ce3+ cation. Scanning Electron Microscopy (SEM) studies showed that the mechanism of inhibition of the Pr3+ ion is also similar to that of the Ce3+ ion. Potentiodynamic polarization experiments after 30 min immersion time showed greatest suppression of oxygen reduction reaction in neutral chloride solutions (pH 7), which reached a maximum at a Zn2+ ion concentration of 5 mM. Anodic polarization experiments after 30 min immersion time, showed no anodic inhibition by the inhibitor in any concentration (0.1 mM - 10 mM) and at any pH. However, anodic polarization of samples immersed after longer immersion times (upto 4 days) in mildly acidic Zn2+ (pH 4) solutions showed significant reduction in anodic kinetics indicating that zinc also acts as a “slow anodic inhibitor”. In contrast to the polarization experiments, coupons exposed to inhibited acidic solutions at pH 4 showed complete suppression of dissolution of Al2CuMg particles compared to zinc-free solutions in the SEM studies. Samples exposed in pH 4 Zn2+-bearing solution exhibited highest polarization resistance which was also observed to increase with time. In deaerated solutions, the inhibition by Zn2+ at pH 4 is not observed as strongly. The ability to make the interfacial electrolyte

  15. Inhibition of calcium carbonate deposition on stainless steel using olive leaf extract as a green inhibitor. (United States)

    Aidoud, Roumaissa; Kahoul, Abdelkrim; Naamoune, Farid


    The antiscale properties of the aqueous extract of olive (Olea europaea L.) leaves as a natural scale inhibitor for stainless steel surface in Hammam raw water were investigated using chronoamperometry (CA) and electrochemical impedance spectroscopy techniques in conjunction with a microscopic examination. The X-ray diffraction analysis reveals that the scale deposited over the pipe walls consists of pure CaCO3 calcite. The CA, in accordance with electrochemical impedance spectra and scanning electron microscopy, shows that the inhibition efficiency increases with increasing extract concentration. This efficiency is considerably reduced as the temperature is increased.

  16. Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition. (United States)

    Headey, Stephen J; Macaskill, Ursula K; Wright, Michele A; Claridge, Jolyon K; Edwards, Patrick J B; Farley, Peter C; Christeller, John T; Laing, William A; Pascal, Steven M


    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

  17. Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition (United States)

    Headey, Stephen J.; MacAskill, Ursula K.; Wright, Michele A.; Claridge, Jolyon K.; Edwards, Patrick J. B.; Farley, Peter C.; Christeller, John T.; Laing, William A.; Pascal, Steven M.


    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin. PMID:20538608

  18. Inhibition of Sunn pest, Eurygaster integriceps, α-amylases by α-amylase inhibitors (T-αAI) from Triticale. (United States)

    Mehrabadi, Mohammad; Bandani, Ali R; Saadati, Fatemeh


    The effect of triticale α-amylases inhibitors on starch hydrolysis catalyzed by the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) midgut amylases was examined. Biochemical studgawies showed that inhibitors from Triticale (a hybrid of wheat and rye) had inhibitiory effects on E. integriceps α-amylases. The effects of the triticale α-amylase inhibitor (T-αAI) on α-amylase of E. integriceps showed a dose dependent manner of inhibition, e.g. less inhibition of enzyme activity (around 10%) with a lower dose (0.25 mg protein) and high inhibition of enzyme activity (around 80%) when a high dose of inhibitor was used (1.5 mg protein). The enzyme kinetic studies using Michaelis-Menten and Lineweaver-Burk equations showed the K(m) remained constant (0.58%) but the maximum velocity (V(max)) decreased in the presence of a crude extract of Triticale inhibitors, indicating mixed inhibition. The temperature giving 50% inactivation of enzyme (T(50)) during a 30-min incubation at pH 7.0 was 73° C. The maximum inhibitory activity was achieved at 35° C and pH 5.0. Gel assays showed the meaningful inhibition of E. integriceps α-amylases by various concentrations of Triticale inhibitors. Based on the data presented in this study, it could be said that the T-αAI has good inhibitory activity on E. integriceps gut α-amylase.

  19. Cargo and dynamin regulate clathrin-coated pit maturation.

    Directory of Open Access Journals (Sweden)

    Dinah Loerke


    Full Text Available Total internal reflection fluorescence microscopy (TIR-FM has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs. To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.

  20. Bacterial dynamin as a membrane puncture repair kit

    NARCIS (Netherlands)

    de Sousa Borges, Anabela; Scheffers, Dirk-Jan


    Dynamin(-like) proteins (DLPs) are widely distributed inbacteria, and both structural and biochemical data haveclearly demonstrated that, like their eukaryotic counter-parts, these proteins can function in membrane modelling.Although various functions for bacterial DLPs have beenhypothesized, a clea

  1. Kinetics of Phosphatase of Regenerating Liver-3 (PRL-3) Inhibition by Small-molecular Inhibitors

    Institute of Scientific and Technical Information of China (English)


    Phosphatase of Regenerating Liver-3 (PRL-3) is a newly identified colorectal cancer metastasis-related protein,which isa 22 kDa non-classical protein tyrosine phosphatase with a C-terminal prenylation motif. In this study, the inhibition kinetics of protein tyrosine phosphatases (PTPs) by a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) was evaluated. PRL-3 exhibits classical Michaelis-Menten kinetics with a vmax value of the inhibitor magnolol can cause Km to increase, but does not alter the vmax value, which suggests the competitive inhibition of PRL-3. At the same time, it was found that DiFMUP is a more sensitive substrate for PRL-3 than para-nitrophenyl phosphate(pNPP) that is more frequently used at present. Furthermore, the method of screening for PTPs by the use of DiFMUP was developed, which studied the acceptance of DiFMUP by other PTPs.

  2. Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death.

    Directory of Open Access Journals (Sweden)

    Pieter J A de Koning

    Full Text Available Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4 M(-1 s(-1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.

  3. Experimental study on scale inhibition performance of a green scale inhibitor polyaspartic acid

    Institute of Scientific and Technical Information of China (English)


    Static and dynamic experiments were carried out to validate scale inhibition performance of a green scale inhibitor-polyaspartic acid (PASP). From the static experiment, it was shown that below 60℃, polyaspartic acid is very effective in scale inhibition, with the scale inhibition ratio exceeding 90% with only 3 mg/L PASP for the 600 mg/L hardness solution. For a higher hardness solution of 800 mg/L, the scale inhibition ratio can also reach 90% with 6 and 12 mg/L PASP at 30 and 60℃respectively. The SEM photographs of CaCO3 crystals indicate that the crystal structure transforms from a compact stick-shape to a loose shape so that the scale can be washed away easily instead of being deposited on the heat transfer surface. The dynamic experimental results show that almost no scales formed on the heat trans- fer surface and the fouling thermal resistance decreases extraordinarily if PASP is added in the solution.

  4. Time-dependent inhibition of CYP3A4 by sertraline, a selective serotonin reuptake inhibitor. (United States)

    Masubuchi, Yasuhiro; Kawaguchi, Yuki


    Drug-drug interactions associated with selective serotonin reuptake inhibitors (SSRIs) are widely known. A major interaction by SSRIs is the inhibition of cytochrome P450 (P450)-mediated hepatic drug metabolism. The SSRI, sertraline, is also reported to increase the blood concentration of co-administered drugs. The potency of sertraline directly to inhibit hepatic drug metabolism is relatively weak compared with the other SSRIs, implying that additional mechanisms are involved in the interactions. The study examined whether sertraline produces time-dependent inhibition of CYP3A4 and/or other P450 enzymes. Incubation of human liver microsomes with sertraline in the presence of NADPH resulted in marked decreases in testosterone 6β-hydroxylation activities, indicating that sertraline metabolism leads to CYP3A4 inactivation. This inactivation required NADPH and was not protected by glutathione. No significant inactivation was observed for other P450 enzymes. Spectroscopic evaluation revealed that microsomes with and without sertraline in the presence of NADPH gave a Soret peak at 455 nm, suggesting the formation of metabolic intermediate (MI) complexes of sertraline metabolite(s) with the reduced form of P450. This is the first report indicating that sertraline produced time-dependent inhibition of CYP3A4, which may be associated with MI complex formation.

  5. Experimental study on scale inhibition performance of a green scale inhibitor polyaspartic acid

    Institute of Scientific and Technical Information of China (English)

    QUAN ZhenHua; CHEN YongChang; WANG XiuRong; SHI Cheng; LIU YunJie; MA ChongFang


    Static and dynamic experiments were carried out to validate scale inhibition performance of a green scale inhibitor-polyaspartic acid (PASP). From the static experiment, it was shown that below 60℃, polyaspartic acid is very effective in scale inhibition, with the scale inhibition ratio exceeding 90% with only 3 mg/L PASP for the 600 mg/L hardness solution. For a higher hardness solution of 800 mg/L, the scale inhibition ratio can also reach 90% with 6 and 12 mg/L PASP at 30 and 60℃ respectively. The SEM photographs of CaCO3 crystals indicate that the crystal structure transforms from a compact stick-shape to a loose shape so that the scale can be washed away easily instead of being deposited on the heat transfer surface. The dynamic experimental results show that almost no scales formed on the heat trans-fer surface and the fouling thermal resistance decreases extraordinarily if PASP is added in the solution.

  6. Factor VII activating protease (FSAP) promotes the proteolysis and inhibition of tissue factor pathway inhibitor (TFPI) (United States)

    Kanse, Sandip M.; Declerck, Paul J.; Ruf, Wolfram; Broze, George; Etscheid, Michael


    Objectives Factor VII activating protease (FSAP) activates FVII as well as pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating haemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk for carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Results and Methods Using screening procedures to determine the influence of FSAP on various haemostasis-related processes on endothelial cells we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anti-coagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP, that specifically inhibited FSAP binding to TFPI, reversed the inhibitory effect of FSAP on TFPI. Conclusions The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating haemostasis and proliferative remodeling events in the vasculature. PMID:22116096

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

    Sheffield, William P; Bhakta, Varsha


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

  8. Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange (United States)

    van Schie, Karin A.; Ooijevaar-de Heer, Pleuni; Dijk, Lisanne; Kruithof, Simone; Wolbink, Gertjan; Rispens, Theo


    Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab. PMID:27605058

  9. Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase. (United States)

    Ndongwe, Tanyaradzwa P; Adedeji, Adeyemi O; Michailidis, Eleftherios; Ong, Yee Tsuey; Hachiya, Atsuko; Marchand, Bruno; Ryan, Emily M; Rai, Devendra K; Kirby, Karen A; Whatley, Angela S; Burke, Donald H; Johnson, Marc; Ding, Shilei; Zheng, Yi-Min; Liu, Shan-Lu; Kodama, Ei-Ichi; Delviks-Frankenberry, Krista A; Pathak, Vinay K; Mitsuya, Hiroaki; Parniak, Michael A; Singh, Kamalendra; Sarafianos, Stefan G


    We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

  10. A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance (United States)

    Lee, Hee Seung; Park, Soo Been; Kim, Sun A; Kwon, Sool Ki; Cha, Hyunju; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Song, Si Young


    Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future. PMID:28134290

  11. Inhibition of hydrate formation by kinetic inhibitors. Literature study; Inhibierung von Erdgashydraten durch kinetische Inhibitoren. Literaturstudie

    Energy Technology Data Exchange (ETDEWEB)

    Eberhardt, E.; Meyn, V.; Rahimian, I. [Institut fuer Erdoel- und Erdgasforschung, Clausthal-Zellerfeld (Germany)


    The aim of this study was to represent the state-of-the art of the inhibition of gas hydrates. Corresponding to recent publications the kinetic inhibition was considered in particular. Special inhibitors were validated using a set of criteria derived from different experimental test methods. Best results were obtained by the application of terpolymer VC-713 especially in relation to nucleation and crystal growth, followed by PVCap (polyvinylcaprolactame) and THI (threshold hydrate inhibitor), the chemical structure of which is derived from the antifreeze glycopeptids of antarcitc winter flounder. (orig.) [German] Die vorliegende Literaturstudie gibt den derzeitigen Stand der Kenntnis zur Inhibierung von Gashydraten wieder. Entsprechend der neueren Literatur wird insbesondere auf die kinetische Inhibierung eingegangen. Zur Beurteilung der verschiedenen Inhibitoren werden Bewertungskriterien zur Validierung der mit unterschiedlichen Untersuchungsmethoden erzielten experimentellen Ergebnisse angegeben. Anhand dieser Vorgehensweise zeigte sich, dass mit dem Terpolymer VC-713 die besten Ergebnisse, insbesondere im Hinblick auf Keimbildung und Wachstum, erzielt werden konnten. Sehr gute Ergebnisse wurden auch mit dem Polyvinylcaprolactam (PVCap) und den aus den Antigefrierpeptiden der antarktischen Winterflunder abgeleiteten Threshold Hydrate Inhibitoren (THI) erhalten. (orig.)

  12. Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice. (United States)

    Xiang, Wei; Ke, Zhiyuan; Zhang, Yong; Cheng, Grace Ho-Yuet; Irwan, Ishak Darryl; Sulochana, K N; Potturi, Padma; Wang, Zhengyuan; Yang, He; Wang, Jingyu; Zhuo, Lang; Kini, R Manjunatha; Ge, Ruowen


    Anti-angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain-hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion-associated domain in MUC4 and other proteins (AMOP) domain at the C-terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C-terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)-basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF-stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase-dependent pathway. ISM binds to αvβ(5) integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmental vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.

  13. [A novel HIF-1 inhibitor--manassantin A derivative LXY6099 inhibits tumor growth]. (United States)

    Lai, Fang-Fang; Liu, Xiao-Yu; Niu, Fei; Lang, Li-Wei; Xie, Ping; Chen, Xiao-Guang


    Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor on hypoxia responses in mammalian tissues. HIF-1 plays as a positive factor in solid tumor and leads to hypoxia-driven responses that enhance its downstream gene expression for tumor growth and survival. LXY6099 was obtained by the structural modification and optimization of manassantin A (MA) as a high potent HIF-1 inhibitor. Antitumor activity of LXY6099 was observed in this study. LXY6099 with an IC50 value of 2.46 x 10(-10) mol x L(-1) showed more sensitive inhibition activity to HIF-1 than that of MA detected by reporter gene assay (> 100 folds). It showed strong inhibition on the growth of human solid tumor cell lines. Furthermore, LXY6099 exhibited significant antitumor activity against established human tumor xenografts in nu/nu mice with treatment of MX-1 breast cancer. Thus, LXY6099 as a novel HIF-1 inhibitor could be further developed into anti-cancer agents.

  14. Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy. (United States)

    Chang, Yu-Wang; Chang, Ya-Ting; Wang, Qinchuan; Lin, Jim Jung-Ching; Chen, Yu-Ju; Chen, Chien-Chang


    Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

  15. Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer. (United States)

    Melisi, Davide; Caputo, Rosa; Damiano, Vincenzo; Bianco, Roberto; Veneziani, Bianca Maria; Bianco, A Raffaele; De Placido, Sabino; Ciardiello, Fortunato; Tortora, Giampaolo


    Biphosphonates (BPs) are widely used to inhibit osteoclastic activity in malignant diseases such as bone metastatic breast and prostate carcinoma. Recent studies reported that BPs could also cause a direct antitumor effect, probably due to their ability to interfere with several intracellular signalling molecules. The enzyme cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) play an important role in the control of cancer cell growth and inhibitors of COX-2 and EGFR have shown antitumor activity in vitro and in vivo in several tumor types. We, and others, have previously shown that EGFR and COX-2 may be directly related to each other and that their selective inhibitors may have a cooperative effect. In the present study we have evaluated the combined effect of zoledronic acid, the most potent nitrogen-containing BP, with the COX-2 inhibitor SC-236 and the selective EGFR-tyrosine kinase inhibitor gefitinib, on breast and prostate cancer models in vitro and in xenografted nude mice. We show that combination of zoledronic acid with SC-236 and gefitinib causes a cooperative antitumor effect accompanied by induction of apoptosis and regulation of the expression of mitogenic factors, proangiogenic factors and cell cycle controllers both in vitro and in xenografted nude mice. The modulatory effect on protein expression and the inhibitory effect on tumor growth is much more potent when the three agents are used together. Since studies are ongoing to explore the antitumor effect of zoledronic acid, our results provide new insights into the mechanism of action of these agents and a novel rationale to translate this feasible combination treatment strategy into a clinical setting.

  16. Disruption of microtubule network rescues aberrant actin comets in dynamin2-depleted cells.

    Directory of Open Access Journals (Sweden)

    Yuji Henmi

    Full Text Available A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.

  17. Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts ▿ † (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C.; Baron, Roland


    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2. PMID:19380485

  18. Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts. (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C; Baron, Roland


    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.

  19. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Honma, Yuichi; Harada, Masaru, E-mail:


    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  20. MEK inhibitor PD98059 acutely inhibits synchronized spontaneous Ca2+ oscillations in cultured hippocampal networks

    Institute of Scientific and Technical Information of China (English)

    Yan-fang RUI; Zhao-hui SUN; Jia-ping GU; Zhong-hua SHENG; Xiang-ping HE; Zuo-ping XIE


    Aim: To investigate the changes in synchronized spontaneous Ca2+ oscillations induced by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 at different concentrations in cultured hippocampal network. Methods: Hippocampal neurons in culture for 1-2 weeks were used for this study. Spontaneous synaptic activities of these hippocampal neurons were examined by Ca2+ imaging using calcium-sensitive dye. MEK inhibitor PD98059 (10,30, and 60 μmol/L) and SB202474 (10 and 60 μmol/L), a negative control for mitogen-activated protein kinase (MAPK) cascade study, were applied to the cells under the microscope while imaging was taking place. Results: PD98059 at a lower concentration of 10 μmol/L had little effect on the Ca2+ oscillation. At the higher concentration of 30 μmol/L, 5 min after application of PD98059, the spike frequency was decreased to 25.38%±7.40% (mean±SEM, n=16, F<0.01 vs medium control) of that of the control period. At an even higher concentration of 60 μmol/L, 5 min after application of PD98059, the spike frequency was decreased to 14.53%±5.34% (mean±SEM, n=16, P<0.01 vs medium control) of that of the control period. The spike amplitude underwent a corresponding decrease. However, the negative control SB202474 at concentrations of 10 and 60 μmol/L had little inhibition effect on the Ca2+ oscillation. Conclusion: These results indicate that PD98059 inhibits synchronized spontaneous Ca2+ oscillation through inhibition of MEK, which hints that the MAPK cascade is required to maintain synchronized spontaneous Ca2+ oscillation.

  1. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages (United States)

    Rossi, A; Pergola, C; Koeberle, A; Hoffmann, M; Dehm, F; Bramanti, P; Cuzzocrea, S; Werz, O; Sautebin, L


    BACKGROUND AND PURPOSE Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo. EXPERIMENTAL APPROACH Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model. KEY RESULTS Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes. CONCLUSIONS AND IMPLICATION Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed. PMID:20880396

  2. BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling (United States)

    Vin, Harina; Ojeda, Sandra S; Ching, Grace; Leung, Marco L; Chitsazzadeh, Vida; Dwyer, David W; Adelmann, Charles H; Restrepo, Monica; Richards, Kristen N; Stewart, Larissa R; Du, Lili; Ferguson, Scarlett B; Chakravarti, Deepavali; Ehrenreiter, Karin; Baccarini, Manuela; Ruggieri, Rosamaria; Curry, Jonathan L; Kim, Kevin B; Ciurea, Ana M; Duvic, Madeleine; Prieto, Victor G; Ullrich, Stephen E; Dalby, Kevin N; Flores, Elsa R; Tsai, Kenneth Y


    Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies. DOI: PMID:24192036

  3. Inhibition of coronary thrombosis and local inflammation by a noncarbohydrate selectin inhibitor. (United States)

    Zoldhelyi, P; Beck, P J; Bjercke, R J; Ober, J C; Hu, X; McNatt, J M; Akhtar, S; Ahmed, M; Clubb, F J; Chen, Z Q; Dixon, R A; Yeh, E T; Willerson, J T


    We tested the hypothesis that selectin inhibition with blocking antibodies or a small-molecular-weight inhibitor of L-, P-, and E-selectin, methoxybenzoylpropionic acid (MBPA), prevents thrombus formation in a canine coronary Folts' model. Cyclic flow variations (CFVs) were induced by crush injury and constriction of the left anterior descending coronary artery in dogs. Systemic infusion of antibodies to P- and L-selectin abolished CFVs, respectively, in 50% and 17% of treated dogs [P = not significant (NS)]. The combination of P- and L-selectin antibodies suppressed CFVs in 60% of treated dogs (P = NS). In contrast, systemic selectin blockade by intravenous infusion or local adventitial application of MBPA markedly reduced CFVs and, in addition, reduced myocardial myeloperoxidase (MPO) activity. We conclude that inhibition of L-, P-, and E-selectin binding by a small-molecular-weight, noncarbohydrate compound markedly reduces arterial thrombosis, whereas systemic administration of antibodies to L- and P-selectin fail to reproduce this antithrombotic effect. These results underscore the role of selectins in the pathogenesis of arterial thrombosis under high shear stress and suggest that inhibition of P- and L- selectin may not suffice to prevent thrombus formation in this model. The role of E-selectin in thrombus formation in this model awaits further testing.

  4. Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor-treated CML stem cells. (United States)

    Zhang, Bin; Chu, Su; Agarwal, Puneet; Campbell, Victoria L; Hopcroft, Lisa; Jørgensen, Heather G; Lin, Allen; Gaal, Karl; Holyoake, Tessa L; Bhatia, Ravi


    Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-κB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML. © 2016 by The American Society of Hematology.

  5. Plasminogen activator inhibitor (PAI)-1 suppresses inhibition of gastric emptying by cholecystokinin (CCK) in mice. (United States)

    Gamble, Joanne; Kenny, Susan; Dockray, Graham J


    The intestinal hormone cholecystokinin (CCK) delays gastric emptying and inhibits food intake by actions on vagal afferent neurons. Recent studies suggest plasminogen activator inhibitor (PAI)-1 suppresses the effect of CCK on food intake. In this study we asked whether PAI-1 also modulated CCK effects on gastric emptying. Five minute gastric emptying of liquid test meals was studied in conscious wild type mice (C57BL/6) and in transgenic mice over-expressing PAI-1 in gastric parietal cells (PAI-1H/Kβ mice), or null for PAI-1. The effects of exogenous PAI-1 and CCK8s on gastric emptying were studied after ip administration. Intragastric peptone delayed gastric emptying in C57BL/6 mice by a mechanism sensitive to the CCK-1 receptor antagonist lorglumide. Peptone did not delay gastric emptying in PAI-1-H/Kβ mice. Exogenous CCK delayed gastric emptying of a control test meal in C57BL/6 mice and this was attenuated by administration of PAI-1; exogenous CCK had no effect on emptying in PAI-1-H/Kβ mice. Prior administration of gastrin to increase gastric PAI-1 inhibited CCK-dependent effects on gastric emptying in C57BL/6 mice but not in PAI-1 null mice. Thus, both endogenous and exogenous PAI-1 inhibit the effects of CCK (whether exogenous or endogenous) on gastric emptying. The data are compatible with emerging evidence that gastric PAI-1 modulates vagal effects of CCK.

  6. In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. (United States)

    Scott, H V; Gero, A M; O'Sullivan, W J


    The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C.

  7. The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated herpesvirus replication in vitro. (United States)

    Gantt, Soren; Carlsson, Jacquelyn; Ikoma, Minako; Gachelet, Eliora; Gray, Matthew; Geballe, Adam P; Corey, Lawrence; Casper, Corey; Lagunoff, Michael; Vieira, Jeffrey


    Kaposi's sarcoma (KS) is the most common HIV-associated cancer worldwide and is associated with high levels of morbidity and mortality in some regions. Antiretroviral (ARV) combination regimens have had mixed results for KS progression and resolution. Anecdotal case reports suggest that protease inhibitors (PIs) may have effects against KS that are independent of their effect on HIV infection. As such, we evaluated whether PIs or other ARVs directly inhibit replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the gammaherpesvirus that causes KS. Among a broad panel of ARVs tested, only the PI nelfinavir consistently displayed potent inhibitory activity against KSHV in vitro as demonstrated by an efficient quantitative assay for infectious KSHV using a recombinant virus, rKSHV.294, which expresses the secreted alkaline phosphatase. This inhibitory activity of nelfinavir against KSHV replication was confirmed using virus derived from a second primary effusion lymphoma cell line. Nelfinavir was similarly found to inhibit in vitro replication of an alphaherpesvirus (herpes simplex virus) and a betaherpesvirus (human cytomegalovirus). No activity was observed with nelfinavir against vaccinia virus or adenovirus. Nelfinavir may provide unique benefits for the prevention or treatment of HIV-associated KS and potentially other human herpesviruses by direct inhibition of replication.

  8. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor. (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min


    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development.

  9. Dynamic modeling of cytochrome P450 inhibition in vitro: impact of inhibitor depletion on IC₅₀ shift. (United States)

    Berry, Loren M; Zhao, Zhiyang; Lin, Min-Hwa Jasmine


    The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.

  10. Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli. (United States)

    Theodorou, Evaggelos C; Theodorou, Marina C; Kyriakidis, Dimitrios A


    AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.

  11. A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis. (United States)

    Venkatasubramanian, Sambasivan; Dhiman, Rohan; Paidipally, Padmaja; Cheekatla, Satyanarayana S; Tripathi, Deepak; Welch, Elwyn; Tvinnereim, Amy R; Jones, Brenda; Theodorescu, Dan; Barnes, Peter F; Vankayalapati, Ramakrishna


    In this study, we found that a subpopulation of CD4(+)CD25(+) (85% Foxp3(+)) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4(+)CD25(+) (85% Foxp3(+)) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+C)D25(+)Foxp3(+)D4GDI(+) cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1β, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+)) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+)CD25(+) (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

  12. A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Sambasivan Venkatasubramanian


    Full Text Available In this study, we found that a subpopulation of CD4(+CD25(+ (85% Foxp3(+ cells from persons with latent tuberculosis infection (LTBI inhibits growth of M. tuberculosis (M. tb in human monocyte-derived macrophages (MDMs. A soluble factor, Rho GDP dissociation inhibitor (D4GDI, produced by apoptotic CD4(+CD25(+ (85% Foxp3(+ cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4(+CD25(+Foxp3(+D4GDI(+ cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1β, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3(+ cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4(+CD25(+ (85% Foxp3+ cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

  13. Oral administration of FAK inhibitor TAE226 inhibits the progression of peritoneal dissemination of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Hao, Hui-fang [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Takaoka, Munenori [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Bao, Xiao-hong [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Wang, Zhi-gang [College of Life Science, Inner Mongolia University, The Key Laboratory of Mammal Reproductive Biology and Biotechnology, Ministry of Education, Hohhot 010021 (China); Tomono, Yasuko [Division of Molecular and Cell Biology, Shigei Medical Research Institute, 2117 Yamada, Okayama 700-0202 (Japan); Sakurama, Kazufumi; Ohara, Toshiaki [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Fukazawa, Takuya; Yamatsuji, Tomoki [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan); Fujiwara, Toshiyoshi [Department of Gastroenterological Surgery, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Kita-ku, Okayama 700-8558 (Japan); Naomoto, Yoshio, E-mail: [Department of General Surgery, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505 (Japan)


    Highlights: Black-Right-Pointing-Pointer A novel FAK inhibitor TAE226 suppressed FAK activity in HCT116 colon cancer cells. Black-Right-Pointing-Pointer TAE226 suppressed proliferation and migration, with a modest effect on adhesion. Black-Right-Pointing-Pointer Silencing of FAK by siRNA made no obvious difference on cancer cell attachment. Black-Right-Pointing-Pointer TAE226 treatment suppressed the progression of peritoneal dissemination. Black-Right-Pointing-Pointer Oral administration of TAE226 prolonged the survival of tumor-bearing mice. -- Abstract: Peritoneal dissemination is one of the most terrible types of colorectal cancer progression. Focal adhesion kinase (FAK) plays a crucial role in the biological processes of cancer, such as cell attachment, migration, proliferation and survival, all of which are essential for the progression of peritoneal dissemination. Since we and other groups have reported that the inhibition of FAK activity exhibited a potent anticancer effect in several cancer models, we hypothesized that TAE226, a novel ATP-competitive tyrosine kinase inhibitor designed to target FAK, can prevent the occurrence and progression of peritoneal dissemination. In vitro, TAE226 greatly inhibited the proliferation and migration of HCT116 colon cancer cells, while their adhesion on the matrix surface was minimally inhibited when FAK activity and expression was suppressed by TAE226 and siRNA. In vivo, when HCT116 cells were intraperitoneally inoculated in mice, the cells could attach to the peritoneum and begin to grow within 24 h regardless of the pretreatment of cells with TAE226 or FAK-siRNA, suggesting that FAK is not essential, at least for the initial integrin-matrix contact. Interestingly, the treatment of mice before and after inoculation significantly suppressed cell attachment to the peritoneum. Furthermore, oral administration of TAE226 greatly reduced the size of disseminated tumors and prolonged survival in tumor-bearing mice. Taken

  14. (R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus. (United States)

    Ivanov, A; Tyzio, R; Zilberter, Y; Ben-Ari, Yehezkel


    Pharmacological agents that mediate a persistent GABAergic conductance are of considerable interest for treatment of epilepsy. (R)-roscovitine is a membrane permeable cyclin-dependent kinase inhibitor, designed to block cell division. It is currently undergoing a phase II clinical trial as an anticancer drug. We show that (R)-roscovitine increases a tonic GABA-mediated current in rat hippocampal neurons. This enhanced tonic current appears independent of synaptic GABA release and requires functional transmembrane GABA transport. The effect of (R)-roscovitine is associated with neither modification of GABAA receptors nor protein kinase activity, but is associated with a significant increase in intracellular GABA concentration in hippocampal GABAergic neurons. (R)-roscovitine-induced tonic inhibition significantly suppresses spontaneous spiking activity of hippocampal pyramidal cells. Therefore, (R)-roscovitine is a potent modulator of neuronal activity in rat hippocampus and may provide a tool for preventing paroxysmal activity.

  15. Caspase Inhibitors may Attenuate Opioid-induced Hyperalgesia and Tolerance via Inhibiting Microglial Activation and Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Jiancheng Zhang


    Full Text Available Prolonged exposure to an opioid induces hyperalgesia and tolerance, which negatively affect pain management in turn and significantly hamper the application of opioids. A growing body of evidence has demonstrated that glial activation contributes to the development of these two side effects. Recent studies have demonstrated that morphine, binding to an accessory protein of Toll-like receptor 4 (TLR4, activates microglia and produces neuroinflammation in amanner parallel to lipopolysaccharide. Meanwhile, lipopolysaccharide activates microglia through TLR4/caspase signalling. Therefore, we hypothesise that morphine may activate microglia throughTLR4/caspase signalling and that caspase inhibitors may attenuate opioid-induced hyperalgesia and tolerance via inhibiting microglial activation and neuroinflammation

  16. Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases. (United States)

    Willenbrock, Frances; Thomas, Daniel A; Amour, Augustin


    Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

  17. Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages

    Directory of Open Access Journals (Sweden)

    Persidsky Yuri


    Full Text Available Abstract Background Lipopolysaccharide (LPS, the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS contributes to neuronal injury. Bowman-Birk inhibitor (BBI, a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures. Methods Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA oxidation. Cytokine expression was determined by quantitative real-time PCR. Results LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α and of ROS. In contrast, BBI pretreatment (1-100 μg/ml of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml, had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml had no effect on N-methyl-D-aspartic acid (NMDA-mediated neurotoxicity. Conclusions These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

  18. Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles. (United States)

    Gheldof, Damien; Mullier, François; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian


    Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.


    Directory of Open Access Journals (Sweden)



    Full Text Available The kinetic characteristics, of interaction between hK6 (human Kallikrein and soybean (BBI , protease inhibitor and antitumor agent , in the presence of substrate (Phenylalanine –Serine-Arginine-(7-amino-4- methyl-coumarin (FSR-AMC were investigated. The hK6 were found to bind soybean in two reversible steps, by slow binding inhibition mechanism. The Ki of the first step binding was 13 nM and Ki* of the second binding step was 1.6 nM. The microcopic rate constants were calculated as follows: 311 M-1.S-1 for k3 , 0.04×10-6 M-1.S-1 for k –3 , 0.2×10-6 S-1 for k 4 and 0.025×10-6 S-1 for k-4 respectively.The results suggested that the interaction mechanism between hK6 and soybean was like that of trypsin with this inhibitor but with rather lower inhibitory constants values.

  20. Inhibition of Pig Phosphoenolpyruvate Carboxykinase Isoenzymes by 3-Mercaptopicolinic Acid and Novel Inhibitors (United States)

    Hidalgo, Jorge; Latorre, Pedro; Carrodeguas, José Alberto; Velázquez-Campoy, Adrián; Sancho, Javier; López-Buesa, Pascual


    There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C. PMID:27391465

  1. Glycolytic inhibitors 2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration. (United States)

    Feng, Xiaolan; Wang, Pan; Liu, Quanhong; Zhang, Ting; Mai, Bingjie; Wang, Xiaobing


    Most cancer cells have the specially increased glycolytic phenotype, which makes this pathway become an attractive therapeutic target. Although glycolytic inhibitor 2-deoxyglucose (2-DG) has been demonstrated to potentiate the cytotoxicity of photodynamic therapy (PDT), the impacts on cell migration after the combined treatment has never been reported yet. The present study aimed to analyze the influence of glycolytic inhibitors 2-DG and 3-bromopyruvate (3-BP) combined with Ce6-PDT on cell motility of Triple Negative Breast Cancer MDA-MB-231 cells. As determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium-bromide-Tetraz-olium (MTT) assay, more decreased cell viability was observed in 2-DG + PDT and 3-BP + PDT groups when compared with either monotherapy. Under optimal conditions, synergistic potentiation on cell membrane destruction and the decline of cell adhesion and cells migratory ability were observed in both 2-DG + PDT and 3-BP + PDT by electron microscope observation (SEM), wound healing and trans-well assays. Besides, serious microfilament network collapses as well as impairment of matrix metalloproteinases-9 (MMP-9) were notably improved after the combined treatments by immunofluorescent staining. These results suggest that 2-DG and 3-BP can both significantly potentiated Ce6-PDT efficacy of cell migration inhibition.

  2. Inhibition of leukemic cells by valproic acid, an HDAC inhibitor, in xenograft tumors (United States)

    Zhang, Zhihua; Hao, Changlai; Wang, Lihong; Liu, Peng; Zhao, Lei; Zhu, Cuimin; Tian, Xia


    The chimeric fusion protein, AML1-ETO, generated by translocation of t(8;21), abnormally recruits histone deacetylase (HDAC) to the promoters of AML1 target genes, resulting in transcriptional repression of the target genes and development of t(8;21) acute myeloid leukemia. Abnormal expression of cyclin-dependent kinase inhibitors, especially p21, is considered a possible mechanism of the arrested maturation and differentiation seen in leukemia cells. A new generation of HDAC inhibitors is becoming an increasing focus of attention for their ability to induce differentiation and apoptosis in tumor cells and to block the cell cycle. Our previous research had demonstrated that valproic acid induces G0/G1 arrest of Kasumi-1 cells in t(8;21) acute myeloid leukemia. In this study, we further confirmed that valproic acid inhibits the growth of Kasumi-1 cells in a murine xenograft tumor model, and that this occurs via upregulation of histone acetylation in the p21 promoter region, enhancement of p21 expression, suppression of phosphorylation of retinoblastoma protein, blocking of transcription activated by E2F, and induction of G0/G1 arrest. PMID:23836985

  3. Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts ▿ †



    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and ...

  4. Common Membrane Trafficking Defects of Disease Associated Dynamin 2 Mutations


    Liu, Ya-Wen; Lukiyanchuk, Vasyl; Schmid, Sandra L.


    Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin-mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, Centronuclear Myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease-associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue-specific diseases. Thus, although the cell...

  5. SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity.

    Directory of Open Access Journals (Sweden)

    Sai Krishnan

    Full Text Available Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3 domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD. Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.

  6. Sonic hedgehog inhibitors prevent colitis-associated cancer via orchestrated mechanisms of IL-6/gp130 inhibition, 15-PGDH induction, Bcl-2 abrogation, and tumorsphere inhibition. (United States)

    Kangwan, Napapan; Kim, Yoon-Jae; Han, Young Min; Jeong, Migyeong; Park, Jong-Min; Go, Eun-Jin; Hahm, Ki-Baik


    Sonic hedgehog (SHH) signaling is essential in normal development of the gastrointestinal (GI) tract, whereas aberrantly activated SHH is implicated in GI cancers because it facilitates carcinogenesis by redirecting stem cells. Since colitis-associated cancer (CAC) is associated with inflammatory bowel diseases, in which SHH and IL-6 signaling, inflammation propagation, and cancer stem cell (CSC) activation have been implicated, we hypothesized that SHH inhibitors may prevent CAC by blocking the above SHH-related carcinogenic pathways. In the intestinal epithelial cells IEC-6 and colon cancer cells HCT-116, IL-6 expression and its signaling were assessed with SHH inhibitors and levels of other inflammatory mediators, proliferation, apoptosis, tumorsphere formation, and tumorigenesis were also measured. CAC was induced in C57BL/6 mice by administration of azoxymethane followed by dextran sodium sulfate administration. SHH inhibitors were administered by oral gavage and the mice were sacrificed at 16 weeks. TNF-α-stimulated IEC-6 cells exhibited increased levels of proinflammatory cytokines and enzymes, whereas SHH inhibitors suppressed TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 signaling. SHH inhibitors significantly induced apoptosis, inhibited cell proliferation, suppressed tumorsphere formation, and reduced stemness factors. In the mouse model, SHH inhibitors significantly reduced tumor incidence and multiplicity, decreased the expression of IL-6, TNF-α, COX-2, STAT3, and NF-κB, and significantly induced apoptosis. In colosphere xenografts, SHH inhibitor significantly suppressed tumorigenesis by inhibiting tumorsphere formation. Taken together, our data suggest that administration of SHH inhibitors could be an effective strategy to prevent colitis-induced colorectal carcinogenesis, mainly by targeting IL-6 signaling, ablating CSCs, and suppressing oncogenic inflammation, achieving chemoquiescence ultimately.

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

    Directory of Open Access Journals (Sweden)

    Minyong Kang


    Full Text Available 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

  8. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition. (United States)

    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D; Nencioni, Alessio


    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

  9. HIV nonnucleoside reverse transcriptase inhibitors and trimethoprim-sulfamethoxazole inhibit plasmodium liver stages. (United States)

    Hobbs, Charlotte V; Voza, Tatiana; De La Vega, Patricia; Vanvliet, Jillian; Conteh, Solomon; Penzak, Scott R; Fay, Michael P; Anders, Nicole; Ilmet, Tiina; Li, Yonghua; Borkowsky, William; Krzych, Urszula; Duffy, Patrick E; Sinnis, Photini


    Although nonnucleoside reverse transcriptase inhibitors (NNRTIs) are usually part of first-line treatment regimens for human immunodeficiency virus (HIV), their activity on Plasmodium liver stages remains unexplored. Additionally, trimethoprim-sulfamethoxazole (TMP-SMX), used for opportunistic infection prophylaxis in HIV-exposed infants and HIV-infected patients, reduces clinical episodes of malaria; however, TMP-SMX effect on Plasmodium liver stages requires further study. We characterized NNRTI and TMP-SMX effects on Plasmodium liver stages in vivo using Plasmodium yoelii. On the basis of these results, we conducted in vitro studies assessing TMP-SMX effects on the rodent parasites P. yoelii and Plasmodium berghei and on the human malaria parasite Plasmodium falciparum. Our data showed NNRTI treatment modestly reduced P. yoelii liver stage parasite burden and minimally extended prepatent period. TMP-SMX administration significantly reduced liver stage parasite burden, preventing development of patent parasitemia in vivo. TMP-SMX inhibited development of rodent and P. falciparum liver stage parasites in vitro. NNRTIs modestly affect liver stage Plasmodium parasites, whereas TMP-SMX prevents patent parasitemia. Because drugs that inhibit liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts. Understanding HIV drug effects on Plasmodium liver stages will aid in optimizing treatment regimens for HIV-exposed and HIV-infected infected patients in malaria-endemic areas.

  10. Cyclooxygenase-2 inhibitor, celecoxib, inhibits leiomyoma cell proliferation through the nuclear factor κB pathway. (United States)

    Park, Seung Bin; Jee, Byung Chul; Kim, Seok Hyun; Cho, Yeon Jean; Han, Myoungseok


    Our aim was to investigate whether celecoxib, a cyclooxygenase 2 (COX-2) inhibitor, decreases the in vitro proliferation of leiomyoma cells if the inflammatory pathway is blocked. Menstruation is an inflammation of uterus that produces cytokines and prostanoids, but the inflammatory mechanism underlying the growth of leiomyoma remains unexplained. Using in vitro cultures of leiomyoma cells obtained from 5 patients who underwent hysterectomy, cell proliferation, inflammatory signaling, transcription factors, growth factors, and extracellular matrix were examined by (4,5-dimethylthiaxol-2-yi)-2,5-diphenyltetraxolium bromide assay, immunoblotting, and quantitative polymerase chain reaction. Prostaglandin E2 was used to induce menstruation-like condition in the cells. We found that celecoxib inhibited COX-2 through the expression of nuclear factor κB in the cells. Celcoxib also decreased the gene expression of interleukin 6, tumor necrosis factor α, collagen A, fibronectin, platelet-derived growth factor, epidermal growth factor, and transforming growth factor β. In conclusion, the present study indicated that celecoxib could inhibit leiomyoma cell proliferation through blocking the inflammatory pathway that is probably one of the mechanisms underlying its pathogenesis.

  11. The PI3K inhibitor GS-1101 synergistically potentiates HDAC inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and ERK pathways (United States)

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T.; Portell, Craig A.; Lannutti, Brian J.; Almasan, Alexandru; Hsi, Eric D.


    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines and primary Non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic. PMID:23889282

  12. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)


    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  13. Inhibition of electrocatalytic O(2) reduction of functional CcO models by competitive, non-competitive, and mixed inhibitors. (United States)

    Collman, James P; Dey, Abhishek; Barile, Christopher J; Ghosh, Somdatta; Decréau, Richard A


    Electrocatalytic reduction of O(2) by functional cytochrome C Oxidase (CcO) models is studied in the presence of several known inhibitors like CO, N(3)(-), CN(-), and NO(2)(-). These models successfully reproduce the inhibitions observed in CcO at similar concentrations reported for these inhibitors. Importantly, the data show very different electrochemical responses depending on the nature of the inhibitor, that is, competitive, non-competitive and mixed. Chemical models have been provided for these observed differences in the electrochemical behavior. Using the benchmark electrochemical behaviors for known inhibitors, the inhibition by NO(2)(-) is investigated. Electrochemical data suggests that NO(2)(-) acts as a competitive inhibitor at high concentrations. Spectroscopic data suggests that NO released during oxidation of the reduced catalyst in presence of excess NO(2)(-) is the source of the competitive inhibition by NO(2)(-). Presence of the distal Cu(B) lowers the inhibitory effect of CN(-) and NO(2)(-). While for CN(-) it weakens its binding affinity to the reduced complex by approximately 4.5 times, for NO(2)(-), it allows regeneration of the active catalyst from a catalytically inactive, air stable ferrous nitrosyl complex via a proposed superoxide mediated pathway.

  14. Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling.

    Directory of Open Access Journals (Sweden)

    Makoto Kondo

    Full Text Available To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.

  15. Rituximab inhibits structural joint damage in patients with rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitor therapies

    NARCIS (Netherlands)

    Keystone, E.; Emery, P.; Peterfy, C.G.; Tak, P.P.; Cohen, S.; Genovese, M.C.; Dougados, M.; Burmester, G.R.; Greenwald, M.; Kvien, T.K.; Williams, S.; Hagerty, D.; Cravets, M.W.; Shaw, T.


    OBJECTIVE: To determine if treatment with a B cell-targeted therapy can inhibit the progression of structural joint damage in patients with rheumatoid arthritis (RA), exhibiting an inadequate response to tumour necrosis factor (TNF) inhibitors. METHODS: In this phase III study, patients with an inad

  16. Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA. (United States)

    Pollet, Annick; Sansen, Stefaan; Raedschelders, Gert; Gebruers, Kurt; Rabijns, Anja; Delcour, Jan A; Courtin, Christophe M


    Triticum aestivum xylanase inhibitor (TAXI)-type inhibitors are active against microbial xylanases from glycoside hydrolase family 11, but the inhibition strength and the specificity towards different xylanases differ between TAXI isoforms. Mutational and biochemical analyses of TAXI-I, TAXI-IIA and Bacillus subtilis xylanase A showed that inhibition strength and specificity depend on the identity of only a few key residues of inhibitor and xylanase [Fierens K et al. (2005) FEBS J 272, 5872-5882; Raedschelders G et al. (2005) Biochem Biophys Res Commun335, 512-522; Sorensen JF & Sibbesen O (2006) Protein Eng Des Sel 19, 205-210; Bourgois TM et al. (2007) J Biotechnol 130, 95-105]. Crystallographic analysis of the structures of TAXI-IA and TAXI-IIA in complex with glycoside hydrolase family 11 B. subtilis xylanase A now provides a substantial explanation for these observations and a detailed insight into the structural determinants for inhibition strength and specificity. Structures of the xylanaseinhibitor complexes show that inhibition is established by loop interactions with active-site residues and substrate-mimicking contacts in the binding subsites. The interaction of residues Leu292 of TAXI-IA and Pro294 of TAXI-IIA with the -2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity. Also, detailed analysis of the interaction interfaces of the complexes illustrates that the inhibition strength of TAXI is related to the presence of an aspartate or asparagine residue adjacent to the acid/base catalyst of the xylanase, and therefore to the pH optimum of the xylanase. The lower the pH optimum of the xylanase, the stronger will be the interaction between enzyme and inhibitor, and the stronger the resulting inhibition.

  17. Inhibiting Properties of Morpholine as Corrosion Inhibitor for Mild Steel in 2N Sulphuric Acid and Phosphoric Acid Medium

    Directory of Open Access Journals (Sweden)

    K. Jayanthi


    Full Text Available The inhibition effect of morpholine on the corrosion of mild steel in 2N sulphuric acid and phosphoric acid has been studied by mass loss and polarization techniques between 302K and 333K. The inhibition efficiency increased with increase in concentration. The corrosion rate increased with increase in temperature and decreased with increase in concentration of inhibitor compared to blank. The adsorption of inhibitor on the mild steel surface has been found to obey Temkin's adsorption isotherm. Potentiostatic polarization results reveal that morpholine act as mixed type inhibitor. The values of activation energy (Ea, free energy of adsorption (∆Gads, enthalpy of adsorption (∆H, and entropy of adsorption (∆S were also calculated.

  18. Nitric oxide synthase inhibitors improve prepulse inhibition responses of Wistar rats. (United States)

    Issy, A C; Lazzarini, M; Szawka, R E; Carolino, R O G; Anselmo-Franci, J A; Del Bel, E A


    Cognitive and attentional deficits in schizophrenia include impairment of the sensorimotor filter as measured by prepulse inhibition (PPI). In this way, the study of animals that naturally present low PPI responses could be a useful approach for screening new antipsychotic drugs. Several pieces of evidence suggest that dopamine and nitric oxide (NO) can modulate PPI but their role in those animals is unknown. The aim of this study was to investigate the role of dopamine and NO in Wistar rats with naturally low PPI response. Male Wistar rats with low PPI responses received an i.p. injection of the antipsychotics haloperidol (0.1, 0.3 or 1mg/kg) or clozapine (0.5, 1.5 or 5mg/kg), the anxiolytic diazepam (1 or 3mg/kg) or the NO synthase (NOS) inhibitors, N(G)- nitro-l-arginine (l-NOARG; 40mg/kg, acutely or sub-chronically) or 7-Nitroindazole (7-NI; 3, 10 or 30mg/kg). All animals were submitted to the PPI test 1h after injection. Striatal and cortical dopamine, DOPAC, and noradrenaline levels of rats with low PPI responses were compared to rats with normal PPI responses. We found increased levels of catecholamines on the striatum and prefrontal cortex of Wistar rats with low PPI. In these animals, both antipsychotics, typical and atypical, and NOS inhibitors significantly increased PPI. Taken together, our findings suggest that the low PPI phenotype may be driven by an overactive catecholamine system. Additionally, our results corroborate the hypothesis of dopamine and NO interaction on PPI modulation and suggest that Wistar rats with low PPI may represent an interesting non-pharmacological model to evaluate new potential antipsychotics. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Inhibition of leukemic cells by valproic acid, an HDAC inhibitor, in xenograft tumors

    Directory of Open Access Journals (Sweden)

    Zhang Z


    Full Text Available Zhihua Zhang,1 Changlai Hao,1 Lihong Wang,1 Peng Liu,2 Lei Zhao,1 Cuimin Zhu,1 Xia Tian31Hematology Department, Affiliated Hospital of Chengde Medical College, Chengde, Hebei Province, 2Department of Medical Oncology, Shijiazhuang Municipal No 1 Hospital, Hebei Province, 3Department of Medical Oncology, Rizhao Municipal People’s Hospital, Shandong Province, People's Republic of ChinaAbstract: The chimeric fusion protein, AML1-ETO, generated by translocation of t(8;21, abnormally recruits histone deacetylase (HDAC to the promoters of AML1 target genes, resulting in transcriptional repression of the target genes and development of t(8;21 acute myeloid leukemia. Abnormal expression of cyclin-dependent kinase inhibitors, especially p21, is considered a possible mechanism of the arrested maturation and differentiation seen in leukemia cells. A new generation of HDAC inhibitors is becoming an increasing focus of attention for their ability to induce differentiation and apoptosis in tumor cells and to block the cell cycle. Our previous research had demonstrated that valproic acid induces G0/G1 arrest of Kasumi-1 cells in t(8;21 acute myeloid leukemia. In this study, we further confirmed that valproic acid inhibits the growth of Kasumi-1 cells in a murine xenograft tumor model, and that this occurs via upregulation of histone acetylation in the p21 promoter region, enhancement of p21 expression, suppression of phosphorylation of retinoblastoma protein, blocking of transcription activated by E2F, and induction of G0/G1 arrest.Keywords: valproic acid, acute myeloid leukemia, AML1-ETO, p21, E2F

  20. Kinetics of Corrosion Inhibition of Aluminum in Acidic Media by Water-Soluble Natural Polymeric Pectates as Anionic Polyelectrolyte Inhibitors

    Directory of Open Access Journals (Sweden)

    Refat M. Hassan


    Full Text Available Corrosion inhibition of aluminum (Al in hydrochloric acid by anionic polyeletrolyte pectates (PEC as a water-soluble natural polymer polysaccharide has been studied using both gasometric and weight loss techniques. The results drawn from these two techniques are comparable and exhibit negligible differences. The inhibition efficiency was found to increase with increasing inhibitor concentration and decrease with increasing temperature. The inhibition action of PEC on Al metal surface was found to obey the Freundlich isotherm. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated and a suitable corrosion mechanism consistent with the kinetic results is discussed in the paper.

  1. Synergistic inhibition of colon carcinoma cell growth by Hedgehog-Gli1 inhibitor arsenic trioxide and phosphoinositide 3-kinase inhibitor LY294002. (United States)

    Cai, Xinyi; Yu, Kun; Zhang, Lijuan; Li, Yunfeng; Li, Qiang; Yang, Zhibin; Shen, Tao; Duan, Lincan; Xiong, Wei; Wang, Weiya


    The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

  2. Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

    Directory of Open Access Journals (Sweden)

    Franco A Rossato

    Full Text Available The metabolic enzyme fatty acid synthase (FASN is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD. The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin

  3. Corrosion inhibition mechanisms of aluminum alloy 2024-T3 by selected non-chromate inhibitors (United States)

    Lopez-Garrity, Omar A.

    The pursuit to find a chromate-alternative has led to the development of several chromate-free aerospace primers and coating systems that offer good protection. However, fundamental understanding of the functionality of the chromate-free pigments that are embedded within these coating systems is lacking. The objective of this study was to understand the fundamental mechanism of corrosion inhibition of aluminum alloy 2024-T3 by molybdate (MoO 42-), silicate (SiO32-), and praseodymium (Pr3+) with the goal of developing the kind of understanding that was accomplished for chromate. Furthermore, since most inhibiting conversion coatings and pigments act by releasing soluble species into the local environment, it was of interest to understand the mechanism of inhibition in aqueous 0.1 M NaCl solution. The mechanism of inhibition of AA2024-T3 by the select non-chromate inhibitors was investigated using various electrochemical, microscopic and spectroscopic techniques. Naturally aerated polarization curves showed that molybdate provided mixed inhibition in near-neutral pH and at a threshold concentration of 0.1 M. The largest effect was a 250 mV increase in the breakdown potential associated with pitting and a 350 mV decrease in the open-circuit potential (OCP). In addition, electrochemical impedance indicated that the corrosion inhibition mechanism is oxygen-dependent owing to the protection afforded by Mo(VI) species. It was proposed that the corrosion inhibition of AA2024-T3 by molybdate may occur following a two-step process whereby molybdate is rapidly reduced to MoO.(OH)2 over the intermetallic particles and is subsequently oxidized to intermediate molybdenum oxides (e.g. Mo4O11) in the presence of oxygen which is reduced. This in turn may lead to a local acidification, promoting the condensation and polymerization of molybdate species in solution to form polymolybdate species (Mo7O24 6- and Mo8O264-). Furthermore, S-phase particle dissolution is decreased

  4. The c-Met Inhibitor MSC2156119J Effectively Inhibits Tumor Growth in Liver Cancer Models

    Energy Technology Data Exchange (ETDEWEB)

    Bladt, Friedhelm, E-mail:; Friese-Hamim, Manja; Ihling, Christian; Wilm, Claudia; Blaukat, Andree [EMD Serono, and Merck Serono Research and Development, Merck KGaA, Darmstadt 64293 (Germany)


    The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only high-affinity ligand. Aberrant activation of c-Met is associated with many human malignancies, including hepatocellular carcinoma (HCC). We investigated the in vivo antitumor and antimetastatic efficacy of the c-Met inhibitor MSC2156119J (EMD 1214063) in patient-derived tumor explants. BALB/c nude mice were inoculated with MHCC97H cells or with tumor fragments of 10 patient-derived primary liver cancer explants selected according to c-Met/HGF expression levels. MSC2156119J (10, 30, and 100 mg/kg) and sorafenib (50 mg/kg) were administered orally as single-agent treatment or in combination, with vehicle as control. Tumor response, metastases formation, and alpha fetoprotein (AFP) levels were measured. MSC2156119J inhibited tumor growth and induced complete regression in mice bearing subcutaneous and orthotopic MHCC97H tumors. AFP levels were undetectable after 5 weeks of MSC2156119J treatment, and the number of metastatic lung foci was reduced. Primary liver explant models with strong c-Met/HGF activation showed increased responsiveness to MSC2156119J, with MSC2156119J showing similar or superior activity to sorafenib. Tumors characterized by low c-Met expression were less sensitive to MSC2156119J. MSC2156119J was better tolerated than sorafenib, and combination therapy did not improve efficacy. These findings indicate that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met high-expressing tumors, supporting its clinical development as an antitumor treatment for HCC patients with active c-Met signaling.

  5. Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1

    Directory of Open Access Journals (Sweden)

    Lihu Gong


    Full Text Available Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1 [4] (Fig. 1. Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7–18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1 recombinant expression and purification of a PAI-1 variant (14-1B containing four mutations (N150H, K154T, Q319L, and M354I, and a tPA serine protease domain (tPA-SPD variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering [19]; (2 formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3 solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19,20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].

  6. Hepatocellular Toxicity Associated with Tyrosine Kinase Inhibitors: Mitochondrial Damage and Inhibition of Glycolysis

    Directory of Open Access Journals (Sweden)

    Franziska Paech


    Full Text Available Tyrosine kinase inhibitors (TKIs are anticancer drugs with a lesser toxicity than classical chemotherapeutic agents but still with a narrow therapeutic window. While hepatotoxicity is known for most TKIs, underlying mechanisms remain mostly unclear. We therefore aimed at investigating mechanisms of hepatotoxicity for imatinib, sunitinib, lapatinib and erlotinib in vitro. We treated HepG2 cells, HepaRG cells and mouse liver mitochondria with TKIs (concentrations 1–100 μM for different periods of time and assessed toxicity. In HepG2 cells maintained with glucose (favoring glycolysis, all TKIs showed a time- and concentration-dependent cytotoxicity and, except erlotinib, a drop in intracellular ATP. In the presence of galactose (favoring mitochondrial metabolism, imatinib, sunitinib and erlotinib showed a similar toxicity profile as for glucose whereas lapatinib was less toxic. For imatinib, lapatinib and sunitinib, cytotoxicity increased in HepaRG cells induced with rifampicin, suggesting formation of toxic metabolites. In contrast, erlotinib was more toxic in HepaRG cells under basal than CYP-induced conditions. Imatinib, sunitinib and lapatinib reduced the mitochondrial membrane potential in HepG2 cells and in mouse liver mitochondria. In HepG2 cells, these compounds increased reactive oxygen species production, impaired glycolysis, and induced apoptosis. In addition, imatinib and sunitinib impaired oxygen consumption and activities of complex I and III (only imatinib, and reduced the cellular GSH pool. In conclusion, imatinib and sunitinib are mitochondrial toxicants after acute and long-term exposure and inhibit glycolysis. Lapatinib affected mitochondria only weakly and inhibited glycolysis, whereas the cytotoxicity of erlotinib could not be explained by a mitochondrial mechanism.

  7. Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

    DEFF Research Database (Denmark)

    Chircop, Megan; Sarcevic, Boris; Larsen, Martin Røssel


    was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and GST-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting...

  8. Recombinant human C1-inhibitor inhibits cytotoxicity induced by allo- and xenoantibodies. (United States)

    Poirier, N; Blancho, G


    Antibody-mediated rejection (AMR) is usually poorly controlled, especially in the context of pretransplant immunization, and remains an unsolved issue in xenotransplantation. In order to study prevention and/or treatment of AMR through an early blockade of the complement classical pathway, we designed two strategies to test the effect of a new recombinant human C1-inhibitor that inhibits C1 esterase (rhC1-INH; Pharming, The Netherlands), in a complement-dependent cytotoxicity assay, in the contexts of pretransplant anti-donor alloimmunization and pig-to-primate combinations in order to compare the situations. RhC1-INH appeared to be efficient, in allo- and xenotransplantation settings to block cytotoxicity when given at the initiation of (preventive strategy) or during (curative strategy) the cytotoxicity assay. Importantly, we showed that a small amount of exogenous rhC1-INH was sufficient to prevent cytotoxicity induced by anti-donor alloantibody, thus possibly helping to prevent or treat AMR in preimmunized patients. These in vitro data lead to future in vivo studies in models of AMR in pigs and baboons in allotransplantation and xenotransplantation, in which cytotoxicity due to Gal and non-Gal antibodies is so detrimental.

  9. Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance. (United States)

    Rabi, S Alireza; Laird, Gregory M; Durand, Christine M; Laskey, Sarah; Shan, Liang; Bailey, Justin R; Chioma, Stanley; Moore, Richard D; Siliciano, Robert F


    HIV-1 protease inhibitors (PIs) are among the most effective antiretroviral drugs. They are characterized by highly cooperative dose-response curves that are not explained by current pharmacodynamic theory. An unresolved problem affecting the clinical use of PIs is that patients who fail PI-containing regimens often have virus that lacks protease mutations, in apparent violation of fundamental evolutionary theory. Here, we show that these unresolved issues can be explained through analysis of the effects of PIs on distinct steps in the viral life cycle. We found that PIs do not affect virion release from infected cells but block entry, reverse transcription, and post-reverse transcription steps. The overall dose-response curves could be reconstructed by combining the curves for each step using the Bliss independence principle, showing that independent inhibition of multiple distinct steps in the life cycle generates the highly cooperative dose-response curves that make these drugs uniquely effective. Approximately half of the inhibitory potential of PIs is manifest at the entry step, likely reflecting interactions between the uncleaved Gag and the cytoplasmic tail (CT) of the Env protein. Sequence changes in the CT alone, which are ignored in current clinical tests for PI resistance, conferred PI resistance, providing an explanation for PI failure without resistance.

  10. Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1. (United States)

    Ramer, Robert; Merkord, Jutta; Rohde, Helga; Hinz, Burkhard


    Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB(1) and CB(2) receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness. Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.

  11. Alpha-fluoromethylhistidine, a histamine synthesis inhibitor, inhibits orexin-induced wakefulness in rats. (United States)

    Yasuko, Seki; Atanda, Akanmu Moses; Masato, Matsuura; Kazuhiko, Yanai; Kazuki, Honda


    Orexins A and B are involved in the regulation of feeding and arousal state. Previously, we reported that third intracerebroventricular (icv) infusion of both orexins A and B induced a significant arousal effect in rats. We determined the effects of intraperitoneal (i.p.) injection of alpha-fluoromethylhistidine (alpha-FMH), a histamine synthesis inhibitor, on orexin-induced wakefulness in freely behaving rats. Male Sprague-Dawley rats were chronically implanted with cortical electroencephalogram (EEG) and neck electromyogram (EMG) electrodes, and a cannula for icv infusion. EEG and EMG were monitored for three consecutive days during continuous icv saline infusion at a rate of 10 microl/h. For a 5-h diurnal period, orexin-B (10 nmol/50 microl saline) replaced the icv infusion of saline. alpha-FMH (100mg/kg, i.p.) was administered 6h before icv infusion of orexin-B. Orexin-B at a dose of 10 nmol/h markedly increased the amount of wakefulness by 99.4% (p<0.05) over the baseline value, whereas alpha-FMH decreased orexin-B-induced wakefulness by 48.8%. Orexin-B-induced suppression of non-REM sleep was reversed by alpha-FMH treatment. Pretreatment with alpha-FMH, significantly inhibited orexin-B-induced wakefulness in rats. The findings of this study therefore suggest that arousal-state regulation by orexin neurons is possibly mediated via the histaminergic system in the tuberomammilary nucleus.

  12. Rice GDP dissociation inhibitor 3 inhibits OsMAPK2 activity through physical interaction. (United States)

    Heo, Jae Bok; Yi, Young Byung; Bahk, Jeong Dong


    GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity.

  13. Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor. (United States)

    Assis, Diego Magno; Gontijo, Vanessa Silva; de Oliveira Pereira, Ivan; Santos, Jorge Alexandre Nogueira; Camps, Ihosvany; Nagem, Tanus Jorge; Ellena, Javier; Izidoro, Mario Augusto; dos Santos Tersariol, Ivarne Luis; de Barros, Nilana Meza Tenório; Doriguetto, Antonio Carlos; dos Santos, Marcelo Henrique; Juliano, Maria Aparecida


    Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K(I) of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver-Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C' in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.

  14. Inhibition of Lassa virus and Ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin. (United States)

    Kolokoltsov, Andrey A; Adhikary, Shramika; Garver, Jennifer; Johnson, Lela; Davey, Robert A; Vela, Eric M


    Arenaviruses and filoviruses are capable of causing hemorrhagic fever syndrome in humans. Limited therapeutic and/or prophylactic options are available for humans suffering from viral hemorrhagic fever. In this report, we demonstrate that pre-treatment of host cells with the kinase inhibitors genistein and tyrphostin AG1478 leads to inhibition of infection or transduction in cells infected with Ebola virus, Marburg virus, and Lassa virus. In all, the results demonstrate that a kinase inhibitor cocktail consisting of genistein and tyrphostin AG1478 is a broad-spectrum antiviral that may be used as a therapeutic or prophylactic against arenavirus and filovirus hemorrhagic fever.

  15. Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition. (United States)

    Ding, Xiaohui; Zhang, Xiujuan; Chong, Huihui; Zhu, Yuanmei; Wei, Huamian; Wu, Xiyuan; He, Jinsheng; Wang, Xinquan; He, Yuxian


    The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor

  16. Inhibition of Myeloperoxidase Activity in Cystic Fibrosis Sputum by Peptide Inhibitor of Complement C1 (PIC1) (United States)

    Hair, Pamela S.; Sass, Laura A.; Krishna, Neel K.


    Myeloperoxidase is the major peroxidase enzyme in neutrophil granules and implicated in contributing to inflammatory lung damage in cystic fibrosis. Free myeloperoxidase is present in cystic fibrosis lung fluid and generates hypochlorous acid. Here we report a new inhibitor of myeloperoxidase activity, Peptide Inhibitor of Complement C1 (PIC1). Using TMB as the oxidizing substrate, PIC1 inhibited myeloperoxidase activity in cystic fibrosis sputum soluble fractions by an average of a 3.4-fold decrease (P = 0.02). PIC1 also dose-dependently inhibited myeloperoxidase activity in a neutrophil lysate or purified myeloperoxidase by up to 28-fold (P < 0.001). PIC1 inhibited myeloperoxidase activity similarly, on a molar basis, as the specific myeloperoxidase inhibitor 4-Aminobenzoic acid hydrazide (ABAH) for various oxidizing substrates. PIC1 was able to protect the heme ring of myeloperoxidase from destruction by NaOCl, assayed by spectral analysis. PIC1 incubated with oxidized TMB reversed the oxidation state of TMB, as measured by absorbance at 450 nm, with a 20-fold reduction in oxidized TMB (P = 0.02). This result was consistent with an antioxidant mechanism for PIC1. In summary, PIC1 inhibits the peroxidase activity of myeloperoxidase in CF sputum likely via an antioxidant mechanism. PMID:28135312

  17. CDC25 Inhibition in Acute Myeloid Leukemia–A Study of Patient Heterogeneity and the Effects of Different Inhibitors

    Directory of Open Access Journals (Sweden)

    Annette K. Brenner


    Full Text Available Cell division cycle 25 (CDC25 protein phosphatases regulate cell cycle progression through the activation of cyclin-dependent kinases (CDKs, but they are also involved in chromatin modulation and transcriptional regulation. CDC25 inhibition is regarded as a possible therapeutic strategy for the treatment of human malignancies, including acute myeloid leukemia (AML. We investigated the in vitro effects of CDC25 inhibitors on primary human AML cells derived from 79 unselected patients in suspension cultures. Both the previously well-characterized CDC25 inhibitor NSC95397, as well as five other inhibitors (BN82002 and the novel small molecular compounds ALX1, ALX2, ALX3, and ALX4, only exhibited antiproliferative effects for a subset of patients when tested alone. These antiproliferative effects showed associations with differences in genetic abnormalities and/or AML cell differentiation. However, the responders to CDC25 inhibition could be identified by analysis of global gene expression profiles. The differentially expressed genes were associated with the cytoskeleton, microtubules, and cell signaling. The constitutive release of 28 soluble mediators showed a wide variation among patients and this variation was maintained in the presence of CDC25 inhibition. Finally, NSC95397 had no or only minimal effects on AML cell viability. In conclusion, CDC25 inhibition has antiproliferative effects on primary human AML cells for a subset of patients, and these patients can be identified by gene expression profiling.

  18. The natural compound fucoidan from New Zealand Undaria pinnatifida synergizes with the ERBB inhibitor lapatinib enhancing melanoma growth inhibition. (United States)

    Thakur, Varsha; Lu, Jun; Roscilli, Giuseppe; Aurisicchio, Luigi; Cappelletti, Manuela; Pavoni, Emiliano; White, William Lindsey; Bedogni, Barbara


    Melanoma remains one of the most aggressive and therapy-resistant cancers. Finding new treatments to improve patient outcomes is an ongoing effort. We previously demonstrated that melanoma relies on the activation of ERBB signaling, specifically of the ERBB3/ERBB2 cascade. Here we show that melanoma tumor growth is inhibited by 60% over controls when treated with lapatinib, a clinically approved inhibitor of ERBB2/EGFR. Importantly, tumor growth is further inhibited to 85% when the natural compound fucoidan from New Zealand U. pinnatifida is integrated into the treatment regimen. Fucoidan not only enhances tumor growth inhibition, it counteracts the morbidity associated with prolonged lapatinib treatment. Fucoidan doubles the cell killing capacity of lapatinib. These effects are associated with a further decrease in AKT and NFκB signaling, two key pathways involved in melanoma cell survival. Importantly, the enhancing cell killing effects of fucoidan can be recapitulated by inhibiting ERBB3 by either a specific shRNA or a novel, selective ERBB3 neutralizing antibody, reiterating the key roles played by this receptor in melanoma. We therefore propose the use of lapatinib or specific ERBB inhibitors, in combination with fucoidan as a new treatment of melanoma that potentiates the effects of the inhibitors while protecting from their potential side effects.

  19. Functional interaction of Parkinson's disease-associated LRRK2 with members of the dynamin GTPase superfamily (United States)

    Stafa, Klodjan; Tsika, Elpida; Moser, Roger; Musso, Alessandra; Glauser, Liliane; Jones, Amy; Biskup, Saskia; Xiong, Yulan; Bandopadhyay, Rina; Dawson, Valina L.; Dawson, Ted M.; Moore, Darren J.


    Mutations in LRRK2 cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase domains, and putative protein–protein interaction domains. Familial PD mutations alter the GTPase and kinase activity of LRRK2 in vitro. LRRK2 is suggested to regulate a number of cellular pathways although the underlying mechanisms are poorly understood. To explore such mechanisms, it has proved informative to identify LRRK2-interacting proteins, some of which serve as LRRK2 kinase substrates. Here, we identify common interactions of LRRK2 with members of the dynamin GTPase superfamily. LRRK2 interacts with dynamin 1–3 that mediate membrane scission in clathrin-mediated endocytosis and with dynamin-related proteins that mediate mitochondrial fission (Drp1) and fusion (mitofusins and OPA1). LRRK2 partially co-localizes with endosomal dynamin-1 or with mitofusins and OPA1 at mitochondrial membranes. The subcellular distribution and oligomeric complexes of dynamin GTPases are not altered by modulating LRRK2 in mouse brain, whereas mature OPA1 levels are reduced in G2019S PD brains. LRRK2 enhances mitofusin-1 GTP binding, whereas dynamin-1 and OPA1 serve as modest substrates of LRRK2-mediated phosphorylation in vitro. While dynamin GTPase orthologs are not required for LRRK2-induced toxicity in yeast, LRRK2 functionally interacts with dynamin-1 and mitofusin-1 in cultured neurons. LRRK2 attenuates neurite shortening induced by dynamin-1 by reducing its levels, whereas LRRK2 rescues impaired neurite outgrowth induced by mitofusin-1 potentially by reversing excessive mitochondrial fusion. Our study elucidates novel functional interactions of LRRK2 with dynamin-superfamily GTPases that implicate LRRK2 in the regulation of membrane dynamics important for endocytosis and mitochondrial morphology. PMID:24282027

  20. Balancing spatially regulated β-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers. (United States)

    Cruz, Lissette A; Vedula, Pavan; Gutierrez, Natasha; Shah, Neel; Rodriguez, Steven; Ayee, Brian; Davis, Justin; Rodriguez, Alexis J


    Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized β-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and β-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed β-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated β-actin translation and dynamin

  1. Organic compounds as corrosion inhibitors for mild steel in acidic media: correlation between inhibition efficiency and chemical structure

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Elizandra C.S.; Chrisman, Erika C.A.N. [Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ (Brazil). Escola de Quimica


    The use of inhibitors for mild steels corrosion control which are in contact with aggressive environment is an accepted practice in acid treatment of oil-wells. Organic compounds have been studied to evaluate their corrosion inhibition potential. Film-forming corrosion inhibitors, commonly used to protect oil-field equipment, can be absorbed on the steel surface to give structurally ordered layers. Therefore, the electrons should act as an important role for this adsorption. Studies reveal that organic compounds show significant inhibition efficiency. For this purpose, their molecules should contain N, O and S heteroatoms in various functional groups, long hydrocarbon linear or branched radical and anion and cation active components. However, most of these compounds are not only expensive but also toxic to living beings. According to the 'Green Chemistry' rules, corrosion inhibitors based on organic compounds should be cheap, with low toxicity and have high inhibition efficiency. In this study, the effects of some organic compounds with different groups such as amide, ether, phenyldiamine, anime and aminophenol on the corrosion behavior of mild steel in acidic media have been investigated. The experimental data were obtained by gravimetric measurements. The results show that these compounds reveal a promising corrosion inhibition where phenyldiamine is the most efficient. The effect of molecular structure on the corrosion inhibition efficiency was investigated by semi-empirical quantum chemical calculations. The electronic properties such as highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO) energy levels, and LUMO-HOMO energy gap orbital density were calculated. The relations between the inhibition efficiency and some quantum parameters are discussed and correlations are proposed. The highest values for the HOMO densities were found in the vicinity nitrogen atom, indicating that it is the most probable adsorption center

  2. Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Zhengzhi, E-mail: [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Luo, Xiaoyong [Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, Luoyang 471000 (China); Nie, Peipei [KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510000 (China); Wu, Baoyan; Zhang, Tao; Wei, Yanchun [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Wang, Wenyi [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Geng, Guojun; Jiang, Jie [Xiamen Cancer Center, Department of Thoracic Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Mi, Yanjun, E-mail: [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China)


    SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuating AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.

  3. Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor. (United States)

    Todd, M J; Hausinger, R P


    Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.

  4. mTOR inhibitors block Kaposi sarcoma growth by inhibiting essential autocrine growth factors and tumor angiogenesis. (United States)

    Roy, Debasmita; Sin, Sang-Hoon; Lucas, Amy; Venkataramanan, Raman; Wang, Ling; Eason, Anthony; Chavakula, Veenadhari; Hilton, Isaac B; Tamburro, Kristen M; Damania, Blossom; Dittmer, Dirk P


    Kaposi sarcoma originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi sarcoma-associated herpesvirus (KSHV) are endemic, Kaposi sarcoma is the most common cancer overall, but model systems for disease study are insufficient. Here, we report the development of a novel mouse model of Kaposi sarcoma, where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, and RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results show that mTOR inhibitors exert a direct anti-Kaposi sarcoma effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for Kaposi sarcoma and other cancers of endothelial origin.

  5. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei. (United States)

    Turrens, J F; Bickar, D; Lehninger, A L


    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  6. Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram; Chen, Albert H.; Dolinski, Brian; Xu, Youyuan; Keilhack, Heike; Nguyen, Thi; Wiznerowicz, Maciej; Li, Lixia; Lutterbach, Bart A.; Chi, An; Paweletz, Cloud; Allison, Timothy; Yan, Youwei; Munshi, Sanjeev K.; Klippel, Anke; Kraus, Manfred; Bobkova, Ekaterina V.; Deshmukh, Sujal; Xu, Zangwei; Mueller, Uwe; Szewczak, Alexander A.; Pan, Bo-Sheng; Richon, Victoria; Pollock, Roy; Blume-Jensen, Peter; Northrup, Alan; Andersen, Jannik N. (Merck)


    Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

  7. Differential Potential of Pharmacological PARP Inhibitors for Inhibiting Cell Proliferation and Inducing Apoptosis in Human Breast Cancer Cells. (United States)

    Węsierska-Gądek, Józefa; Mauritz, Matthias; Mitulovic, Goran; Cupo, Maria


    BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s). © 2015 Wiley Periodicals, Inc.

  8. Structural basis of specific inhibition of tissue-type plasminogen activator by plasminogen activators inhibitor-1 (United States)

    Gong, Lihu; Liu, Min; Zeng, Tu; Shi, Xiaoli; Yuan, Cai; Andreasen, Peter A.; Huang, Mingdong


    Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19]. PMID:26909366

  9. Two dynamin-2 genes are required for normal zebrafish development.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Gibbs

    Full Text Available Dynamin-2 (DNM2 is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.

  10. Dynamin-related proteins in plant post-Golgi traffic

    Directory of Open Access Journals (Sweden)

    Masaru eFujimoto


    Full Text Available Membrane traffic between two organelles begins with the formation of transport vesicles from the donor organelle. Dynamin-related proteins (DRPs, which are large multidomain GTPases, play crucial roles in vesicle formation in post-Golgi traffic. Numerous in vivo and in vitro studies indicate that animal dynamins, which are members of DRP family, assemble into ring- or helix-shaped structures at the neck of a bud site on the donor membrane, where they constrict and sever the neck membrane in a GTP hydrolysis-dependent manner. While much is known about DRP-mediated trafficking in animal cells, little is known about it in plant cells. So far, two structurally distinct subfamilies of plant DRPs (DRP1 and DRP2 have been found to participate in various pathways of post-Golgi traffic. This review summarizes the structural and functional differences between these two DRP subfamilies, focusing on their molecular, cellular and developmental properties. We also discuss the molecular networks underlying the functional machinery centering on these two DRP subfamilies. Furthermore, we hope that this review will provide direction for future studies on the mechanisms of vesicle formation that are not only unique to plants but also common to eukaryotes.

  11. Dynamin-related proteins in plant post-Golgi traffic. (United States)

    Fujimoto, Masaru; Tsutsumi, Nobuhiro


    Membrane traffic between two organelles begins with the formation of transport vesicles from the donor organelle. Dynamin-related proteins (DRPs), which are large multidomain GTPases, play crucial roles in vesicle formation in post-Golgi traffic. Numerous in vivo and in vitro studies indicate that animal dynamins, which are members of DRP family, assemble into ring- or helix-shaped structures at the neck of a bud site on the donor membrane, where they constrict and sever the neck membrane in a GTP hydrolysis-dependent manner. While much is known about DRP-mediated trafficking in animal cells, little is known about it in plant cells. So far, two structurally distinct subfamilies of plant DRPs (DRP1 and DRP2) have been found to participate in various pathways of post-Golgi traffic. This review summarizes the structural and functional differences between these two DRP subfamilies, focusing on their molecular, cellular and developmental properties. We also discuss the molecular networks underlying the functional machinery centering on these two DRP subfamilies. Furthermore, we hope that this review will provide direction for future studies on the mechanisms of vesicle formation that are not only unique to plants but also common to eukaryotes.

  12. Coarse-grained simulation of dynamin-mediated fission (United States)

    Muller, Marcus; Zhang, Guojie; Fuhrmans, Marc

    Fission is a process in which a region of a lipid bilayer is deformed and separated from its host membrane, so that an additional, topologically independent compartment surrounded by a continuous lipid bilayer is formed. It is a fundamental process in the compartmentalization of living organisms and carefully regulated by a number of membrane-shaping proteins. An important group within these is the dynamin family of proteins that are involved in the final severance of the hourglass-shaped neck, via which the growing compartment remains connected to the main volume until the completion of fission. We present computer simulations testing different hypotheses of how dynamin proteins facilitate fission by constriction and curvature. Our results on constraint-induced fission of cylindrical membrane tubes emphasize the importance of the local creation of positive curvature and reveal a complex picture of fission, in which the topological transformation can become arrested in an intermediate stage if the proteins constituting the fission machinery are not adaptive.

  13. Scopolamine induces disruption of latent inhibition which is prevented by antipsychotic drugs and an acetylcholinesterase inhibitor. (United States)

    Barak, Segev; Weiner, Ina


    The fact that muscarinic antagonists may evoke a psychotic state ('antimuscarinic psychosis'), along with findings of cholinergic alterations in schizophrenia, have kindled an interest in the involvement of the cholinergic system in this disorder. Latent inhibition (LI) is a cross-species phenomenon manifested as a poorer conditioning of a stimulus seen when the stage of conditioning is preceded by a stage of repeated nonreinforced pre-exposure to that stimulus, and is considered to index the capacity to ignore irrelevant stimuli. Amphetamine-induced LI disruption and its reversal by antipsychotic drugs (APDs) is a well-established model of positive symptoms of schizophrenia. Here, we tested whether the muscarinic antagonist scopolamine would disrupt LI and whether such disruption would be reversed by APDs and by the acetylcholinesterase inhibitor physostigmine. The results showed that scopolamine at doses of 0.15 and 0.5 mg/kg disrupted LI, and that this effect was due to the action of the drug in the pre-exposure stage, suggesting a role of muscarinic transmission in attentional processes underlying LI. Both the typical and the atypical APDs, haloperidol and clozapine, reversed scopolamine-induced LI disruption when given in conditioning or in both stages, but not in pre-exposure, indicating that the mechanism of antipsychotic action in this model is independent of the mechanism of action of the propsychotic drug. Scopolamine-induced LI disruption was reversed by physostigmine (0.05 and 0.15 mg/kg), which was ineffective in reversing amphetamine-induced LI disruption, pointing to distinct mechanisms underlying LI disruption by these two propsychotic drugs. The latter was further supported by the finding that unlike amphetamine, the LI-disrupting doses of scopolamine did not affect activity levels. We propose scopolamine-induced LI disruption as a model of cholinergic-related positive symptoms in schizophrenia.

  14. Closing escape routes: inhibition of IL-8 signaling enhances the anti-tumor efficacy of PI3K inhibitors. (United States)

    Juvekar, Ashish; Wulf, Gerburg M


    The phosphoinositide 3-kinase (PI3K) pathway serves as a relay where signals that emanate from the cell membrane are received and converted into intracellular signals that promote proliferation and survival. Inhibitors of PI3K hold promise for the treatment of breast cancer because activation of this pathway is highly prevalent. However, as is increasingly observed with inhibitors of cell signaling, there appear to be mechanisms of primary and secondary resistance. Britschgi and colleagues report that compensatory activation of the IL-8 signaling axis is a mechanism of primary resistance to PI3K inhibitors in some triple-negative breast cancers. In a set of experiments that carefully emulate the clinical scenario in a mouse model, they show that simultaneous inhibition of Janus kinase 2 enhances the efficacy of PI3K/mammalian target of rapamycin inhibition. Their paper lends further support to the concept that successful design of treatments with signal transduction inhibitors must anticipate potential escape routes - and include agents to simultaneously block them.

  15. Chickpea (Cicer arietinum) and other plant-derived protease inhibitor concentrates inhibit breast and prostate cancer cell proliferation in vitro. (United States)

    Magee, Pamela J; Owusu-Apenten, Richard; McCann, Mark J; Gill, Chris I; Rowland, Ian R


    The soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), is currently showing great promise as a novel cancer chemopreventive agent. In contrast to the wealth of research conducted on this compound, the anticancer effects of protease inhibitors isolated from other leguminous sources have received limited attention. In the current study, 7 protease inhibitor concentrates (PICs) were isolated from various leguminous sources (including soybean) and characterized. The effects of PICs on the proliferation of breast and prostate cancer cells were investigated in vitro. Chickpea PIC significantly inhibited the viability of MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells at all concentrations tested (25-400 μg/ml). In addition, kidney bean (200, 400 μg/ml), soybean (50, 100 μg/ml), and mungbean (100, 200 μg/ml) PICs inhibited LNCaP cell viability. These findings suggest that leguminous PICs may possess similar anticancer properties to that of soybean BBI and deserve further study as possible chemopreventive agents.

  16. A Revisit to the Corrosion Inhibition of Aluminum in Aqueous Alkaline Solutions by Water-Soluble Alginates and Pectates as Anionic Polyelectrolyte Inhibitors

    Directory of Open Access Journals (Sweden)

    Refat Hassan


    Full Text Available The corrosion behavior of aluminum (Al in alkaline media in presence of some natural polymer inhibitors has been reinvestigated. The inhibition action of the tested inhibitors was found to obey both Langmuir and Freundlich isotherms models. The inhibition efficiency was found to increase with increasing the inhibitors concentration and decrease with increasing the temperature, suggesting physical adsorption mechanism. Factors such as the concentration and geometrical structure of the inhibitor, concentration of the corrosive medium, and temperature affecting the corrosion rates were examined. The kinetic parameters were evaluated, and a suitable corrosion mechanism consistent with the kinetic results obtained is suggested and discussed.

  17. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma


    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D. Ashley; Dome, Jeffrey S.


    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term grow...

  18. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo. (United States)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun; Zhuang, Wen-Fang


    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent.

  19. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity. (United States)

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min


    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

  20. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

    Institute of Scientific and Technical Information of China (English)

    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG


    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  1. G domain dimerization controls dynamin's assembly-stimulated GTPase activity

    Energy Technology Data Exchange (ETDEWEB)

    Chappie, Joshua S.; Acharya, Sharmistha; Leonard, Marilyn; Schmid, Sandra L.; Dyda, Fred (NIH); (Scripps)


    Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin's basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 {angstrom} resolution crystal structure of a human dynamin 1-derived minimal GTPase-GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF{sub 4}{sup -}. The structure reveals dynamin's catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase-GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

  2. Dynamin Forms a Src Kinase–sensitive Complex with Cbl and Regulates Podosomes and Osteoclast Activity (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sanjay, Archana; Horne, William C.; De Camilli, Pietro; Baron, Roland


    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption. PMID:15872089

  3. Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity. (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sanjay, Archana; Horne, William C; De Camilli, Pietro; Baron, Roland


    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.

  4. Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model. (United States)

    Sostelly, Alexandre; Payen, Léa; Guitton, Jérôme; Di Pietro, Attilio; Falson, Pierre; Honorat, Mylène; Boumendjel, Ahcène; Gèze, Annabelle; Freyer, Gilles; Tod, Michel


    ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

  5. Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis.

    Directory of Open Access Journals (Sweden)

    Dipannita Dutta

    Full Text Available Clathrin independent endocytosis (CIE is a form of endocytosis present in all cells that mediates the entry of nutrients, macromolecules and membrane proteins into cells. When compared to clathrin-dependent endocytosis (CDE, however, much less is known about the machinery involved in forming CIE endosomes. One way to distinguish CIE from CDE has been to deplete cells of coat proteins involved in CDE such as clathrin or the dynamin GTPase, leading to a block of CDE but not CIE. A drawback of such genetic manipulations is that depletion of proteins important for mediating CDE over a period of days can have complex indirect effects on cellular function. The identification of chemical compounds that specifically and rapidly block CDE or CIE would facilitate the determination of whether a process involved CDE or CIE. To date, all of those compounds have targeted CDE. Dynasore and the dynoles specifically target and block dynamin activity thus inhibiting CDE but not most forms of CIE. Recently, a new compound called pitstop 2 was identified as an inhibitor of the interaction of amphiphysin with the amino terminal domain of clathrin, and shown to inhibit CDE in cells. Here we show that pitstop 2 is also a potent inhibitor of CIE. The effects of pitstop 2 are not restricted to inhibition of clathrin since knockdown of clathrin fails to rescue the inhibition of endocytosis of CIE proteins by the drug. Thus pitstop 2 has additional cellular targets besides the amino terminal domain of clathrin and thus cannot be used to distinguish CIE from CDE.

  6. A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Marcus J Taylor

    Full Text Available Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.

  7. Clathrin- and dynamin-independent endocytosis of FGFR3--implications for signalling.

    Directory of Open Access Journals (Sweden)

    Ellen Margrethe Haugsten

    Full Text Available Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3 and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.

  8. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis. (United States)

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos


    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  9. Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré


    Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects...... of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50...... correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs...

  10. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse. (United States)

    Baixauli, Francesc; Martín-Cófreces, Noa B; Morlino, Giulia; Carrasco, Yolanda R; Calabia-Linares, Carmen; Veiga, Esteban; Serrador, Juan M; Sánchez-Madrid, Francisco


    During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

  11. A novel dual inhibitor of microtubule and Bruton's tyrosine kinase inhibits survival of multiple myeloma and osteoclastogenesis. (United States)

    Pandey, Manoj K; Gowda, Krishne; Sung, Shen-Shu; Abraham, Thomas; Budak-Alpdogan, Tulin; Talamo, Giampolo; Dovat, Sinisa; Amin, Shantu


    Bruton's tyrosine kinase (BTK) regulates many vital signaling pathways and plays a critical role in cell proliferation, survival, migration, and resistance. Previously, we reported that a small molecule, KS99, is an inhibitor of tubulin polymerization. In the present study, we explored whether KS99 is a dual inhibitor of BTK and tubulin polymerization. Although it is known that BTK is required for clonogenic growth and resistance, and microtubules are essential for cancer cell growth, dual targeting of these two components has not been explored previously. Through docking studies, we predicted that KS99 interacts directly with the catalytic domain of BTK and inhibits phosphorylation at the Y223 residue and kinase activities. Treatment of KS99 reduces the cell viability of multiple myeloma (MM) and CD138(+) cells, with an IC50 of between 0.5 and 1.0 μmol/L. We found that KS99 is able to induce apoptosis in MM cells in a caspase-dependent manner. KS99 suppressed the receptor activator of NF-κB ligand (RANKL)-induced differentiation of macrophages to osteoclasts in a dose-dependent manner and, importantly, inhibited the expression of cytokines associated with bone loss. Finally, we found that KS99 inhibits the in vivo tumor growth of MM cells through the inhibition of BTK and tubulin. Overall, our results show that dual inhibition of BTK and tubulin polymerization by KS99 is a viable option in MM treatment, particularly in the inhibition of refraction and relapse. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  12. Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

    Directory of Open Access Journals (Sweden)

    Jitka Holcakova

    Full Text Available Cyclin-dependent kinases (CDKs are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional

  13. Type II kinase inhibitors show an unexpected inhibition mode against Parkinson's disease-linked LRRK2 mutant G2019S. (United States)

    Liu, Min; Bender, Samantha A; Cuny, Gregory D; Sherman, Woody; Glicksman, Marcie; Ray, Soumya S


    A number of well-known type II inhibitors (ATP-noncompetitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson's disease-linked mutation, G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between the wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work in an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected noncompetitive mechanism against WT. Because the G2019S mutation lies in the DXG motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences. (1) The mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being "locked" into the activated state, and (2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP-competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors because of desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant.

  14. Cysteine based novel noncompetitive inhibitors of urease(s)--distinctive inhibition susceptibility of microbial and plant ureases. (United States)

    Amtul, Zareen; Kausar, Naheed; Follmer, Cristian; Rozmahel, Richard F; Atta-Ur-Rahman; Kazmi, Syed Arif; Shekhani, Mohammed Saleh; Eriksen, Jason L; Khan, Khalid M; Choudhary, Mohammad Iqbal


    Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver-Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum Ki value of 2 microM for N,N-dimethyl L-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.

  15. Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis In Vivo Primarily by Inhibiting VEGF Expression and Angiogenesis

    Directory of Open Access Journals (Sweden)

    Patrick Frost


    Full Text Available We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  16. Identification of proton-pump inhibitor drugs that inhibit Trichomonas vaginalis uridine nucleoside ribohydrolase. (United States)

    Shea, Tara A; Burburan, Paola J; Matubia, Vivian N; Ramcharan, Sandy S; Rosario, Irving; Parkin, David W; Stockman, Brian J


    Trichomonas vaginalis continues to be a major health problem with drug-resistant strains increasing in prevalence. Novel antitrichomonal agents that are mechanistically distinct from current therapies are needed. The NIH Clinical Compound Collection was screened to find inhibitors of the uridine ribohydrolase enzyme required by the parasite to scavenge uracil for its growth. The proton-pump inhibitors omeprazole, pantoprazole, and rabeprazole were identified as inhibitors of this enzyme, with IC50 values ranging from 0.3 to 14.5 μM. This suggests a molecular mechanism for the in vitro antitrichomonal activity of these proton-pump inhibitors, and may provide important insights toward structure-based drug design.

  17. Selective Inhibition of the Synthesis of Sindbis Virion Proteins by an Inhibitor of Chymotrypsin (United States)

    Pfefferkorn, E. R.; Boyle, Mary K.


    Treatment of chick embryo fibroblasts infected with Sindbis virus with TPCK, the choloromethyl ketone derivative of tosyl-phenylalanine and an inhibitor of chymotrypsin, resulted in reduced synthesis of viral structural proteins and the accumulation of a high-molecular-weight polypeptide, thought to be a precursor. The analogous inhibitor of trypsin, TLCK, the chloromethyl ketone derivative of tosyllysine, had no such effect. PMID:5061988

  18. A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition. (United States)

    Agola, Jacob O; Hong, Lin; Surviladze, Zurab; Ursu, Oleg; Waller, Anna; Strouse, J Jacob; Simpson, Denise S; Schroeder, Chad E; Oprea, Tudor I; Golden, Jennifer E; Aubé, Jeffrey; Buranda, Tione; Sklar, Larry A; Wandinger-Ness, Angela


    Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

  19. Molecular modeling study for inhibition mechanism of human chymase and its application in inhibitor design.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymase complexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41 upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most active compound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitory mechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes.

  20. Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

    DEFF Research Database (Denmark)

    Powell, K A; Valova, V A; Malladi, C S


    Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding...... assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine...... and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed...

  1. Membrane-mediated interactions and the dynamics of dynamin oligomers on membrane tubes

    Energy Technology Data Exchange (ETDEWEB)

    Shlomovitz, R; Gov, N S [Department of Chemical Physics, Weizmann Institute of Science, PO Box 26, Rehovot, Israel 76100 (Israel); Roux, A, E-mail: [Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet, CH-1211 Geneva (Switzerland)


    Dynamin is a protein that plays a key role in the transport and recycling of membrane tubes and vesicles within a living cell. This protein adsorbs from solution to PIP{sub 2}-containing membranes, and on these tubes it forms curved oligomers that condense into tight helical domains of uniform radius. The dynamics of this process is treated here in terms of the linear stability of a continuum model, whereby membrane-mediated interactions are shown to drive the spontaneous nucleation of condensed dynamin domains. We furthermore show that the deformation of the membrane outside the dynamin domains induces an energy barrier that can hinder the full coalescence of neighboring growing domains. We compare these calculations to experimental observations on dynamin dynamics in vitro.

  2. Structural basis for mechanochemical role of Arabidopsis thaliana dynamin-related protein in membrane fission

    Institute of Scientific and Technical Information of China (English)

    Liming Yan; Yuanyuan Ma; Yuna Sun; Jian Gao; Xiaoyue Chen; Jiewei Liu; CongwanWang; Zihe Rao; Zhiyong Lou


    Dear Editor Dynamins and dynamin-related proteins (DRPs) constitute a large superfamily of GTPases throughout animal,plant,and bacteria and play essential roles in core cellular processes (Praefcke and McMahon,2004).Plant specific dynamin-related subfamilies share essential functions with those in mammalian cell,e.g.clarthrinmediated endocytosis and fission of mitochondria;yet they also play unique functional roles in plant cells (Hong et al.,2003;Chen et al.,2011;Xue et al.,2011)(Supplementary Figure S1).Key features of dynamin members,including large molecular size,high basal GTP hydrolysis,and self-assembly into filamentous helices,distinguish them from other classical signaling and regulatory GTPases (Praefcke and McMahon,2004).

  3. Discovery of a small molecule PDI inhibitor that inhibits reduction of HIV-1 envelope glycoprotein gp120. (United States)

    Khan, Maola M G; Simizu, Siro; Lai, Ngit Shin; Kawatani, Makoto; Shimizu, Takeshi; Osada, Hiroyuki


    Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.

  4. The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Rajah, T.; Chow, S.C., E-mail:


    The caspase inhibitor benzyloxycarbony (Cbz)-L-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.

  5. Identification of small molecule inhibitors of ERCC1-XPF that inhibit DNA repair and potentiate cisplatin efficacy in cancer cells. (United States)

    Arora, Sanjeevani; Heyza, Joshua; Zhang, Hao; Kalman-Maltese, Vivian; Tillison, Kristin; Floyd, Ashley M; Chalfin, Elaine M; Bepler, Gerold; Patrick, Steve M


    ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease which is essential in multiple DNA repair pathways in mammalian cells. ERCC1-XPF (ERCC1-ERCC4) repairs cisplatin-DNA intrastrand adducts and interstrand crosslinks and its specific inhibition has been shown to enhance cisplatin cytotoxicity in cancer cells. In this study, we describe a high throughput screen (HTS) used to identify small molecules that inhibit the endonuclease activity of ERCC1-XPF. Primary screens identified two compounds that inhibit ERCC1-XPF activity in the nanomolar range. These compounds were validated in secondary screens against two other non-related endonucleases to ensure specificity. Results from these screens were validated using an in vitro gel-based nuclease assay. Electrophoretic mobility shift assays (EMSAs) further show that these compounds do not inhibit the binding of purified ERCC1-XPF to DNA. Next, in lung cancer cells these compounds potentiated cisplatin cytotoxicity and inhibited DNA repair. Structure activity relationship (SAR) studies identified related compounds for one of the original Hits, which also potentiated cisplatin cytotoxicity in cancer cells. Excitingly, dosing with NSC16168 compound potentiated cisplatin antitumor activity in a lung cancer xenograft model. Further development of ERCC1-XPF DNA repair inhibitors is expected to sensitize cancer cells to DNA damage-based chemotherapy.

  6. Regulatory T-Cell Augmentation or Interleukin-17 Inhibition Prevents Calcineurin Inhibitor-Induced Hypertension in Mice. (United States)

    Chiasson, Valorie L; Pakanati, Abhinandan R; Hernandez, Marcos; Young, Kristina J; Bounds, Kelsey R; Mitchell, Brett M


    The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17-producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4(+)/FoxP3(+) regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17-producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor-treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17-neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A-treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice. © 2017 American Heart Association, Inc.

  7. Inhibition of the NOTCH pathway using γ-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors. (United States)

    Dantas-Barbosa, Carmela; Bergthold, Guillaume; Daudigeos-Dubus, Estelle; Blockus, Heike; Boylan, John F; Ferreira, Celine; Puget, Stephanie; Abely, Michel; Vassal, Gilles; Grill, Jacques; Geoerger, Birgit


    Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

  8. Histone deacetylase inhibitors upregulate Rap1GAP and inhibit Rap activity in thyroid tumor cells. (United States)

    Dong, Xiaoyun; Korch, Christopher; Meinkoth, Judy L


    Increases in Rap activity have been associated with tumor progression. Although activating mutations in Rap have not been described, downregulation of Rap1GAP is frequent in human tumors including thyroid carcinomas. In this study, we explored whether endogenous Rap1GAP expression could be restored to thyroid tumor cells. The effects of deacetylase inhibitors and a demethylating agent, individually and in combination, were examined in four differentiated and six anaplastic thyroid carcinoma (ATC) cell lines. Treatment with the structurally distinct histone deacetylase (HDAC) inhibitors, sodium butyrate and trichostatin A, increased Rap1GAP expression in all the differentiated thyroid carcinoma cell lines and in four of the six ATC cell lines. The demethylating agent, 5-aza-deoxycytidine, restored Rap1GAP expression in one anaplastic cell line and enhanced the effects of HDAC inhibitors in a second anaplastic cell line. Western blotting indicated that Rap2 was highly expressed in human thyroid cancer cells. Importantly, treatment with HDAC inhibitors impaired Rap2 activity in both differentiated and anaplastic tumor cell lines. The mechanism through which Rap activity is repressed appears to entail effects on the expression of multiple Rap regulators, including RapGEFs and RapGAPs. These results suggest that HDAC inhibitors may provide a tractable approach to impair Rap activity in human tumor cells.

  9. Cardiovascular risk and inhibition of cyclooxygenase: traditional nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors

    Directory of Open Access Journals (Sweden)

    M. Campanini


    Full Text Available BACKGROUND The development of non-selective nonsteroidal anti-inflammatory drugs (tNSAIDs and, more recently, of selective inhibitors of the cycloooxygenase-2 isoform (COXIBs, has contributed greatly towards the effective management of patients with arthritis and pain complaints. Although COXIBs have demonstrated an improved gastrointestinal tolerability compared with tNSAIDs, the cardiovascular effects of the two drugs types are much controversial. By blocking prostacyclin formation but leaving platelet-derived thromboxane A2 generation unopposed, the potential gastrointestinal benefit of COXIBs may come at cost of increased thrombotic risk. AIM OF THE STUDY This review aims at analysing the cardiovascular effects of the tNSAIDs and COXIBs. METHOD This review addresses the controversy of effects of COXIBs and tNSAIDs in 4 segments. It begins with a discussion about pathophysiological effects of cyclooxygenase inhibition on cardiovascular system. This is followed by a systematic review and meta-analysis of a control, randomized, double blind study and population-based matched case-control study to compare the risk of serious cardiovascular events with tNSAIDs and COXIBs. Then it answers to key questions with the aim to assist the clinicians for a systematic approach to evaluate the risk-benefit-ratio of NSAIDs in the clinical practice. Finally we analyse the open questions associated with the use of NSAIDs and the cardiovascular events. RESULTS The use of rofecoxib demonstrated an increase in adverse cardiovascular events. This toxic effect is not dose-related. The relationship between celecoxib and cardiovascular risk is less clear. The results of different clinical trials are conflicting: some didn’t demonstrate increase in cardiovascular toxicity but the APC study and recently a metanalysis reported a significant incidence of adverse cardiovascular events. Also valdecoxib and parecoxib appear to have increased risk for cardiovascular

  10. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

    Directory of Open Access Journals (Sweden)

    Qureshi Asaf A


    Full Text Available Abstract Background Altered immune function during ageing results in increased production of nitric oxide (NO and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin in lipopolysaccharide (LPS-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. Results The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P 40%; P 60%; P 40%; P P  Conclusions The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli

  11. Recent advances in protein kinase inhibition: current molecular scaffolds used for inhibitor synthesis. (United States)

    Stover, D R; Lydon, N B; Nunes, J J


    Early efforts to discover and develop protein kinase inhibitors have focused largely on a small group of oncology targets such as the EGFR and PKC enzymes. More recently, hundreds of protein kinases have been identified at the genetic level, many of which are now being assigned functions in a variety of signaling pathways. Additionally, mutagenesis and X-ray crystallographic studies have further defined common structural features associated with binding of the ATP cofactor within a conserved ATP binding cleft. These studies have also demonstrated significant differences in the ATP binding cleft between individual kinases, providing a molecular basis for understanding and exploiting inhibitor specificity. The current review will focus on recent developments in the field of ATP site-directed inhibitors with particular emphasis on the major scaffolds being derivatized to take advantage of variable regions of the active site.

  12. The significance of dynamin 2 expression for prostate cancer progression, prognostication, and therapeutic targeting. (United States)

    Xu, Bin; Teng, Liang Hong; Silva, Sabrina Daniela da; Bijian, Krikor; Al Bashir, Samir; Jie, Su; Dolph, Michael; Alaoui-Jamali, Moulay A; Bismar, Tarek A


    Dynamin 2 (Dyn2) is essential for intracellular vesicle formation and trafficking, cytokinesis, and receptor endocytosis. In this study, we investigated the implication of Dyn2 as a prognostic marker and therapeutic target for progressive prostate cancer (PCA). We evaluated Dyn2 protein expression by immunohistochemistry in two cohorts: men with localized PCA treated by retropubic radical prostatectomy (n = 226), and men with advanced/castrate-resistant PCA (CRPC) treated by transurethral resection of prostate (TURP) (n = 253). The role of Dyn2 in cell invasiveness was assessed by in vitro and in vivo experiments using androgen-responsive and refractory PCA preclinical models. Dyn2 expression was significantly increased across advanced stages of PCA compared to benign prostate tissue (P size and lymph node metastases in vivo. In isolated PCA cells, Dyn2 was found to regulate focal adhesion turnover, which is critical for cell migration; this mechanism requires full Dyn2 compared to mutants deficient in GTPase activity. In conclusion, Dyn2 overexpression is associated with neoplastic prostate epithelium and is associated with poor prognosis. Inhibition of Dyn2 prevents cell invasiveness in androgen-responsive and -refractory PCA models, supporting the potential benefit of Dyn2 to serve as a therapeutic target for advanced PCA.

  13. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. (United States)

    Bania, Jacek; Samborski, Jaroslaw; Bogus, Mieczyslawa; Polanowski, Antoni


    The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.

  14. Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: lessons learned from HIV-1 protease inhibition. (United States)

    Shen, Yang; Radhakrishnan, Mala L; Tidor, Bruce


    Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV-1 protease inhibition and the molecular designs targeted a panel of wild-type and drug-resistant mutant HIV-1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders.

  15. Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. (United States)

    Kim, Y-J; Uyama, H


    Tyrosinase is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair. Various dermatological disorders, such as melasma, age spots and sites of actinic damage, arise from the accumulation of an excessive level of epidermal pigmentation. In addition, unfavorable enzymatic browning of plant-derived foods by tyrosinase causes a decrease in nutritional quality and economic loss of food products. The inadequacy of current conventional techniques to prevent tyrosinase action encourages us to seek new potent tyrosinase inhibitors. This article overviews the various inhibitors obtained from natural and synthetic sources with their industrial importance.


    Institute of Scientific and Technical Information of China (English)

    WANG Zhao-Xia; LU Bin-Bin; WANG Teng; YIN Yong-Mei; DE Wei; SHU Yong-Qian


    Background Gene therapy by adenovirus-mediated wild-type p53 gene transfer has been shown to inhibit lung cancer growth in vitro, in animal models, and in human clinical trials. The antitumor effect of selective cyclooxygenase (COX)-2 inhibitors has been demonstrated in preclinical studies. However, no information is available on the effects of p53 gene therapy combined with selective COX-2 inhibitor on COX-2 gene expression and growth inhibition of human lung cancer cells. Methods We evaluated the effects of recombinant adenovirus-p53 (Ad-p53) gene therapy combined with selective COX-2 inhibitor on the proliferation, apoptosis, cell cycle arrest of human lung adenocarcinoma A549 cell line, and the effects of tumor suppressor exogenous wild type p53 on COX-2 gene expression. Results Ad-p53 gene therapy combined with selective COX-2 inhibitor celecoxib shows significant synergistic inhibition effects on the growth of human lung adenocarcinoma A549 cell line. Exogenous p53 gene can suppress COX-2 gene expression. Conclusions Significant synergistic inhibition effects of A549 cell line by the combined Ad-p53 and selective COX-2 inhibitor celecoxib may be achieved by enhancement of growth inhibition, apoptosis induction and suppression of COX-2 gene expression. This study provides first evidence that the administration of p53 gene therapy in combination with COX-2 inhibitors might be a new clinical strategy for the treatment or prevention of NSCLC.

  17. Theoretical study of inhibition efficiencies of some amino acids on corrosion of carbon steel in acidic media: green corrosion inhibitors. (United States)

    Dehdab, Maryam; Shahraki, Mehdi; Habibi-Khorassani, Sayyed Mostafa


    Inhibition efficiencies of three amino acids [tryptophan (B), tyrosine (c), and serine (A)] have been studied as green corrosion inhibitors on corrosion of carbon steel using density functional theory (DFT) method in gas and aqueous phases. Quantum chemical parameters such as EH OMO (highest occupied molecular orbital energy), E LUMO (lowest unoccupied molecular orbital energy), hardness (η), polarizability ([Formula: see text]), total negative charges on atoms (TNC), molecular volume (MV) and total energy (TE) have been calculated at the B3LYP level of theory with 6-311++G** basis set. Consistent with experimental data, theoretical results showed that the order of inhibition efficiency is tryptophan (B) > tyrosine (C) > serine (A). In order to determine the possible sites of nucleophilic and electrophilic attacks, local reactivity has been evaluated through Fukui indices.

  18. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells. (United States)

    Kim, Han Sol; Li, Hongliang; Kim, Hye Won; Shin, Sung Eun; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Bae, Young Min; Park, Won Sun


    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertraline decreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 mu M and a slope value (Hill coefficient) of 0.61. Although the application of 1 mu M sertraline did not affect the steady-state activation curves, sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine, had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From these results, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptake inhibition by shifting inactivation curves to a more negative potential.

  19. The broad-spectrum metalloproteinase inhibitor BB-94 inhibits growth, HER3 and Erk activation in fulvestrant-resistant breast cancer cell lines

    DEFF Research Database (Denmark)

    Kirkegaard, Tove; Yde, Christina Westmose; Kveiborg, Marie


    cells. This was prevented by treatment of resistant cells with the metalloproteinase inhibitor TAPI-2. Only the broad-spectrum metalloproteinase inhibitor BB-94, and not the more selective inhibitors GM6001 or TAPI-2, which inhibited shedding of the HER ligands produced by the fulvestrant...... of ligands. Only the broad-spectrum metalloproteinase inhibitor BB-94 could abrogate HER3 and Erk activation in the resistant cells, which stresses the complexity of the resistance mechanisms and the requirement of targeting signaling from HER receptors by multiple strategies....

  20. The Na+/Glucose Cotransporter Inhibitor Canagliflozin Activates AMPK by Inhibiting Mitochondrial Function and Increasing Cellular AMP Levels. (United States)

    Hawley, Simon A; Ford, Rebecca J; Smith, Brennan K; Gowans, Graeme J; Mancini, Sarah J; Pitt, Ryan D; Day, Emily A; Salt, Ian P; Steinberg, Gregory R; Hardie, D Grahame


    Canagliflozin, dapagliflozin, and empagliflozin, all recently approved for treatment of type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose reuptake by sodium/glucose cotransporter (SGLT) 2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMPK, an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin, or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of acetyl-CoA carboxylase (ACC) 1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue, or spleen. Because phosphorylation of ACC by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared with other SGLT2 inhibitors. © 2016 by the American Diabetes Association.

  1. ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia. (United States)

    Shankar, Deepa B; Li, Junling; Tapang, Paul; Owen McCall, J; Pease, Lori J; Dai, Yujia; Wei, Ru-Qi; Albert, Daniel H; Bouska, Jennifer J; Osterling, Donald J; Guo, Jun; Marcotte, Patrick A; Johnson, Eric F; Soni, Niru; Hartandi, Kresna; Michaelides, Michael R; Davidsen, Steven K; Priceman, Saul J; Chang, Jenny C; Rhodes, Katrin; Shah, Neil; Moore, Theodore B; Sakamoto, Kathleen M; Glaser, Keith B


    In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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} 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.

  3. The class I HDAC inhibitor Romidepsin targets inflammatory breast cancer tumor emboli and synergizes with paclitaxel to inhibit metastasis. (United States)

    Robertson, Fredika M; Chu, Khoi; Boley, Kimberly M; Ye, Zaiming; Liu, Hui; Wright, Moishia C; Moraes, Ricardo; Zhang, Xuejun; Green, Tessa L; Barsky, Sanford H; Heise, Carla; Cristofanilli, Massimo


    Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

  4. Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

    Directory of Open Access Journals (Sweden)

    Deu John M Cruz

    Full Text Available Chikungunya virus (CHIKV is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415, one pyrrolopyridine (CND0545 and one thiazol-carboxamide (CND3514 inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against

  5. Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin II expression.

    Directory of Open Access Journals (Sweden)

    Wen-Chung Tsai

    Full Text Available Low-level laser therapy (LLLT is commonly used to treat sports-related tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. This study was designed to determine the effect of laser on tenocyte migration. Furthermore, the correlation between this effect and expression of dynamin 2, a positive regulator of cell motility, was also investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm with energy density at 1.0, 1.5, and 2.0 J/cm(2. Tenocyte migration was evaluated by an in vitro wound healing model and by transwell filter migration assay. The messenger RNA (mRNA and protein expressions of dynamin 2 were determined by reverse transcription/real-time polymerase chain reaction (real-time PCR and Western blot analysis respectively. Immunofluorescence staining was used to evaluate the dynamin 2 expression in tenocytes. Tenocytes with or without laser irradiation was treated with dynasore, a dynamin competitor and then underwent transwell filter migration assay. In vitro wound model revealed that more tenocytes with laser irradiation migrated across the wound border to the cell-free zone. Transwell filter migration assay confirmed that tenocyte migration was enhanced dose-dependently by laser. Real-time PCR and Western-blot analysis demonstrated that mRNA and protein expressions of dynamin 2 were up-regulated by laser irradiation dose-dependently. Confocal microscopy showed that laser enhanced the expression of dynamin 2 in cytoplasm of tenocytes. The stimulation effect of laser on tenocytes migration was suppressed by dynasore. In conclusion, low-level laser irradiation stimulates tenocyte migration in a process that is mediated by up-regulation of dynamin 2, which can be suppressed by dynasore.

  6. Inhibition of Reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs)



    IAPs comprise a family of inhibitors of apoptosis found in viruses and animals. In vivo binding studies demonstrated that both baculovirus and Drosophila IAPs physically interact with an apoptosis-inducing protein of Drosophila, Reaper (RPR), through their baculovirus IAP repeat (BIR) region. Expression of IAPs blocked RPR-induced apoptosis and resulted in the accumulation of RPR in punctate perinuclear locations which coincided with IAP localization. When expressed alone, RPR rapidly disappe...

  7. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood. (United States)

    Hansson, K M; Nielsen, S; Elg, M; Deinum, J


    Corn trypsin inhibitor (CTI), an inhibitor of FXIIa, is used to prevent plasma coagulation by contact activation, to specifically investigate tissue factor (TF)-initiated coagulation. In the present work the specificity of CTI for factor (F) XIIa is questioned. In the commercial available plasma coagulation assays CTI was found to double activated partial thromboplastin time (APTT) at a plasma concentration of 7.3 ± 1.5 μm CTI (assay concentration 2.4 μm). No effect was found on the prothrombin time (PT) when high TF concentrations were used. Also, with specific antibodies for FXIIa and for FXIa only APTT was found to be extended but not PT. With specific enzyme assays using chromogenic substrates CTI was shown to be a strong inhibitor of FXIIa and a competitive inhibitor of FXIa with Ki  = 8.1 ± 0.3 μm, without effect on the coagulation factors FVIIa, FIXa, FXa and thrombin. In thrombin generation and coagulation (free oscillation rheometry, FOR) assays, initiated with low TF concentrations, no effect of CTI (plasma concentrations of 4.4 and 13.6 μm CTI, 25 resp. 100 mg L(-1) in blood) was found with ≥ 1 pm TF. At ≤ 0.1 pm TF in the FOR whole blood assay the coagulation time (CT) concentration dependently increased while the plasma CT became longer than the observation time. To avoid inhibition of FXIa and the thrombin feedback loop we recommend that for coagulation assays the concentration of CTI in blood should be below 20 mg L(-1) (1.6 μm) and in plasma below 3 μm. © 2014 International Society on Thrombosis and Haemostasis.

  8. JTE-522, a selective COX-2 inhibitor, inhibits growth of pulmonary metastases of colorectal cancer in rats

    Directory of Open Access Journals (Sweden)

    Enomoto Masayuki


    Full Text Available Abstract Background Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model. Methods A suspension of 5 × 106 RCN-9 (rat colon cancer cells was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522 (0, 3, 10, or 30 mg/kg/day was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF. Results JTE-522 dose-dependently decreased lung weight (p = 0.001 and the size of pulmonary metastatic tumors (p = 0.0002. However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors. Conclusion JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer.

  9. Iminosugars as potential inhibitors of glycogenolysis: structural insights into the molecular basis of glycogen phosphorylase inhibition. (United States)

    Oikonomakos, Nikos G; Tiraidis, Costas; Leonidas, Demetres D; Zographos, Spyros E; Kristiansen, Marit; Jessen, Claus U; Nørskov-Lauritsen, Leif; Agius, Loranne


    Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified as potent inhibitors of liver glycogen phosphorylase a (IC(50) = 0.4-1.2 microM) and of basal and glucagon-stimulated glycogenolysis (IC(50) = 1-3 microM). The X-ray structures of 5, 9, and its N-3-phenylpropyl analogue 8 in complex with rabbit muscle glycogen phosphorylase (GPb) shows that iminosugars bind tightly at the catalytic site in the presence of the substrate phosphate and induce conformational changes that characterize the R-state conformation of the enzyme. Charged nitrogen N1 is within hydrogen-bonding distance with the carbonyl oxygen of His377 (5) and in ionic contact with the substrate phosphate oxygen (8 and 9). Our findings suggest that the inhibitors function as oxocarbenium ion transition-state analogues. The conformational change to the R state provides an explanation for previous findings that 5, unlike inhibitors that favor the T state, promotes phosphorylation of GPb in hepatocytes with sequential inactivation of glycogen synthase.

  10. Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs (United States)

    Bianco, R; Garofalo, S; Rosa, R; Damiano, V; Gelardi, T; Daniele, G; Marciano, R; Ciardiello, F; Tortora, G


    Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting. PMID:18319715

  11. A theoretical study on the inhibition efficiencies of some amino acids as corrosion inhibitors of nickel

    Energy Technology Data Exchange (ETDEWEB)

    Gece, Goekhan, E-mail: [Department of Physical Chemistry, Faculty of Science, Ankara University, Besevler, 06100 Ankara (Turkey); Bilgic, Semra [Department of Physical Chemistry, Faculty of Science, Ankara University, Besevler, 06100 Ankara (Turkey)


    To clarify the inhibition efficiencies of a total of 12 amino acids for the corrosion of nickel in acidic medium, a density functional theory (DFT) study was carried out using the B3LYP/LANL2DZ method. Quantum chemical descriptors such as the energy of highest occupied molecular orbital (E{sub HOMO}), energy of lowest unoccupied molecular orbital (E{sub LUMO}), and the energy gap ({Delta}E) were calculated. Equations were proposed using linear regression analysis to determine the most effective parameter on inhibition efficiency. The theoretically obtained results were found to be consistent with the experimental data reported.

  12. The cytoskeletal protein Ndel1 regulates dynamin 2 GTPase activity.

    Directory of Open Access Journals (Sweden)

    Mathieu Chansard

    Full Text Available Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2, a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.

  13. Reduction in dynamin-2 is implicated in ischaemic cardiac arrhythmias. (United States)

    Shi, Dan; Xie, Duanyang; Zhang, Hong; Zhao, Hong; Huang, Jian; Li, Changming; Liu, Yi; Lv, Fei; The, Erlinda; Liu, Yuan; Yuan, Tianyou; Wang, Shiyi; Chen, Jinjin; Pan, Lei; Yu, Zuoren; Liang, Dandan; Zhu, Weidong; Zhang, Yuzhen; Li, Li; Peng, Luying; Li, Jun; Chen, Yi-Han


    Ischaemic cardiac arrhythmias cause a large proportion of sudden cardiac deaths worldwide. The ischaemic arrhythmogenesis is primarily because of the dysfunction and adverse remodelling of sarcolemma ion channels. However, the potential regulators of sarcolemma ion channel turnover and function in ischaemic cardiac arrhythmias remains unknown. Our previous studies indicate that dynamin-2 (DNM2), a cardiac membrane-remodelling GTPase, modulates ion channels membrane trafficking in the cardiomyocytes. Here, we have found that DNM2 plays an important role in acute ischaemic arrhythmias. In rat ventricular tissues and primary cardiomyocytes subjected to acute ischaemic stress, the DNM2 protein and transcription levels were markedly down-regulated. This DNM2 reduction was coupled with severe ventricular arrhythmias. Moreover, we identified that the down-regulation of DNM2 within cardiomyocytes increases the action potential amplitude and prolongs the re-polarization duration by depressing the retrograde trafficking of Nav1.5 and Kir2.1 channels. These effects are likely to account for the DNM2 defect-induced arrhythmogenic potentials. These results suggest that DNM2, with its multi-ion channel targeting properties, could be a promising target for novel antiarrhythmic therapies.

  14. A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice. (United States)

    Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui


    Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel

  15. BRAF kinase inhibitor exerts anti-tumor activity against breast cancer cells via inhibition of FGFR2. (United States)

    Zhang, Zong Xin; Jin, Wen Jun; Yang, Sheng; Ji, Cun Li


    Most anti-angiogenic therapies currently being evaluated in clinical trials targetvascular endothelial growth factor (VEGF) pathway; however, the tumor vasculature can acquire resistance to VEGF-targeted therapy by shifting to other angiogenesis mechanisms. Therefore, other potential therapeutic agents that block non-VEGF angiogenic pathways need to be evaluated. Here we identified BRAF kinase inhibitor, vemurafenibas an agent with potential anti-angiogenic and anti-breast cancer activities. Vemurafenib demonstrated inhibition of endothelial cell proliferation, migration, and tube formation in response to basic fibroblast growth factor (bFGF). In ex vivo and in vivo angiogenesis assays, vemurafenib suppressed bFGF-induced microvessel sprouting of rat aortic rings and angiogenesis in vivo. To understand the underlying molecular basis, we examined the effects of vemurafenib on different molecular components in treated endothelial cell, and found that vemurafenib suppressed bFGF-triggered activation of FGFR2 and protein kinase B (AKT). Moreover, vemurafenib directly inhibited proliferation and blocked the oncogenic signaling pathways in breast cancer cell. In vivo, using xenograft models of breast cancer cells MDA-MB-231, vemurafenib showed growth-inhibitory activity associated with inhibition of tumor angiogenesis. Taken together, our results indicate that vemurafenib targets the FGFR2-mediated AKT signaling pathway in endothelial cells, leading to the suppression of tumor growth and angiogenesis.

  16. Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase Ⅱ inhibitor-induced hepatocellular carcinoma cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Su-jong YU; Hyo-suk LEE; Jung-hwan YOON; Jeong-hoon LEE; Sun-jung MYUNG; Eun-sun JANG; Min-sun KWAK; Eun-ju CHO; Ja-june JANG; Yoon-jun KIM


    Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-Ⅸ (CA-Ⅸ) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-Ⅸ in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients.Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-Ⅸ-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase Ⅱ inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array.Results: Incubation of HCC cells under hypoxia (1% 02, 5% C02, 94% N2) for 36 h significantly increased CA-IX expression level. CAI#1(400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-Ⅸ intensity was inversely related to E-cadherin intensity.Conclusion: Inhibition of hypoxia-induced CA-Ⅸ enhances hexokinase Ⅱ inhibitor-induced HCC apoptosis. Furthermore, CA-IX expres sion profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-Ⅸ, in combination with a hexokinase Ⅱ inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.

  17. Elucidation of clathrin-mediated endocytosis in tetrahymena reveals an evolutionarily convergent recruitment of dynamin.

    Directory of Open Access Journals (Sweden)

    Nels C Elde


    Full Text Available Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1. While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.

  18. Elucidation of clathrin-mediated endocytosis in tetrahymena reveals an evolutionarily convergent recruitment of dynamin.

    Directory of Open Access Journals (Sweden)

    Nels C Elde


    Full Text Available Ciliates, although single-celled organisms, contain numerous subcellular structures and pathways usually associated with metazoans. How this cell biological complexity relates to the evolution of molecular elements is unclear, because features in these cells have been defined mainly at the morphological level. Among these ciliate features are structures resembling clathrin-coated, endocytic pits associated with plasma membrane invaginations called parasomal sacs. The combination of genome-wide sequencing in Tetrahymena thermophila with tools for gene expression and replacement has allowed us to examine this pathway in detail. Here we demonstrate that parasomal sacs are sites of clathrin-dependent endocytosis and that AP-2 localizes to these sites. Unexpectedly, endocytosis in Tetrahymena also involves a protein in the dynamin family, Drp1p (Dynamin-related protein 1. While phylogenetic analysis of AP subunits indicates a primitive origin for clathrin-mediated endocytosis, similar analysis of dynamin-related proteins suggests, strikingly, that the recruitment of dynamin-family proteins to the endocytic pathway occurred independently during the course of the ciliate and metazoan radiations. Consistent with this, our functional analysis suggests that the precise roles of dynamins in endocytosis, as well as the mechanisms of targeting, differ in metazoans and ciliates.

  19. Functional dissection of the dictyostelium discoideum dynamin B mitochondrial targeting sequence.

    Directory of Open Access Journals (Sweden)

    Amrita Rai

    Full Text Available Most mitochondrial proteins are nuclear encoded and synthesized in the cytosol with an N-terminal mitochondrial targeting sequence or presequence for subsequent import into mitochondria. Here, we describe the proteolytic processing and inner membrane potential-dependent translocation of a dynamin family member by the Dictyostelium discoideum mitochondrial import system. Our results show that the unusual D. discoideum dynamin B presequence is removed through a processing mechanism that is common for mitochondrial matrix proteins. We identified a minimal segment of the dynamin B presequence containing seven lysine residues. This 47-residue region is, in combination with consensus matrix protease cleavage sites, necessary and sufficient for mitochondrial targeting. The correct positioning of these lysine residues plays a critical role for the proper processing and mitochondrial import of dynamin B in D. discoideum. Fluorescent proteins tagged with the dynamin B presequence or presequence regions supporting mitochondrial import in D. discoideum are imported with similar efficiency into the mitochondrial matrix of mammalian cells, indicating that the basic mechanisms underlying mitochondrial protein import are highly conserved from amoebozoa to mammalia.

  20. Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts. (United States)

    Eleniste, Pierre P; Du, Liping; Shivanna, Mahesh; Bruzzaniti, Angela


    Bone loss is caused by the dysregulated activity of osteoclasts which degrade the extracellular bone matrix. The tyrosine kinase Pyk2 is highly expressed in osteoclasts, and mice lacking Pyk2 exhibit an increase in bone mass, in part due to impairment of osteoclast function. Pyk2 is activated by phosphorylation at Y402 following integrin activation, but the mechanisms leading to Pyk2 dephosphorylation are poorly understood. In the current study, we examined the mechanism of action of the dynamin GTPase on Pyk2 dephosphorylation. Our studies reveal a novel mechanism for the interaction of Pyk2 with dynamin, which involves the binding of Pyk2's FERM domain with dynamin's plextrin homology domain. In addition, we demonstrate that the dephosphorylation of Pyk2 requires dynamin's GTPase activity and is mediated by the tyrosine phosphatase PTP-PEST. The dephosphorylation of Pyk2 by dynamin and PTP-PEST may be critical for terminating outside-in integrin signaling, and for stabilizing cytoskeletal reorganization during osteoclast bone resorption.

  1. Inhibition of the Proteasome β2 Site Sensitizes Triple-Negative Breast Cancer Cells to β5 Inhibitors and Suppresses Nrf1 Activation. (United States)

    Weyburne, Emily S; Wilkins, Owen M; Sha, Zhe; Williams, David A; Pletnev, Alexandre A; de Bruin, Gerjan; Overkleeft, Hermann S; Goldberg, Alfred L; Cole, Michael D; Kisselev, Alexei F


    The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

  2. Inhibitor Ranking Through QM based Chelation Calculations for Virtual Screening of HIV-1 RNase H inhibition

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Svendsen, Casper Steinmann; Kongsted, Jacob


    Quantum mechanical (QM) calculations have been used to predict the binding affinity of a set of ligands towards HIV-1 RT associated RNase H (RNH). The QM based chelation calculations show improved binding affinity prediction for the inhibitors compared to using an empirical scoring function. Furt....... Thus, the computational models tested in this study could be useful as high throughput filters for searching HIV-1 RNase H active-site molecules in the virtual screening process.......Quantum mechanical (QM) calculations have been used to predict the binding affinity of a set of ligands towards HIV-1 RT associated RNase H (RNH). The QM based chelation calculations show improved binding affinity prediction for the inhibitors compared to using an empirical scoring function...... of the methods based on the use of a training set of molecules, QM based chelation calculations were used as filter in virtual screening of compounds in the ZINC database. By this, we find, compared to regular docking, QM based chelation calculations to significantly reduce the large number of false positives...

  3. A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways (United States)

    Spring, Bryan Q.; Bryan Sears, R.; Zheng, Lei Zak; Mai, Zhiming; Watanabe, Reika; Sherwood, Margaret E.; Schoenfeld, David A.; Pogue, Brian W.; Pereira, Stephen P.; Villa, Elizabeth; Hasan, Tayyaba


    Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)—a multikinase inhibitor—encapsulated inside. Near-infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release while reducing systemic drug exposure and associated toxicities.

  4. Indomethacin but not a selective cyclooxygenase-2 inhibitor inhibits esophageal adenocarcinogenesis in rats

    Institute of Scientific and Technical Information of China (English)

    Paula Esquivias; Antonio Morandeira; Alfredo Escartín; Carmelo Cebrián; Sonia Santander; Francisco Esteva; María Asunción García-González


    AIM:To evaluate the effects of indomethacin [dual cyclooxygenase (COX)-1/COX-2 inhibitor] and 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl) phenyl)-2-(5H)-furanone (MF-tricyclic) (COX-2 selective inhibitor) in a rat experimental model of Barrett's esophagus and esophageal adenocarcinoma.METHODS:A total of 112 surviving post-surgery rats were randomly divided into three groups:the control group (n =48),which did not receive any treatment; the indomethacin group (n =32),which were given 2mg/kg per day of the COX-1/COX-2 inhibitor; and the MF-tricyclic group (n =32),which received 10 mg/kg per day of the selective COX-2 inhibitor.Randomly selected rats were killed either 8 wk or 16 wk after surgery.The timing of the deaths was in accordance with a previous study performed in our group.Only rats that were killed at the times designated by the protocol were included in the study.We then assessed the histology and prostaglandin E2 (PGE2) expression levels in the rat esophagi.An additional group of eight animals that did not undergo esophagojejunostomy were included in order to obtain normal esophageal tissue as a control.RESULTS:Compared to a control group with no treatment (vehicle-treated rats),indomethacin treatment was associated with decreases in ulcerated esophageal mucosa (16% vs 35% and 14% vs 17%,2 mo and 4mo after surgery,respectively; P =0.021),length of intestinal metaplasia in continuity with anastomosis (2± 1.17 mm vs 2.29 ± 0.75 mm and 1.25 ± 0.42 mm vs 3.5 ± 1.54 mm,2 mo and 4 mo after surgery,respectively; P =0.007),presence of intestinal metaplasia beyond anastomosis (20% vs 71.4% and 0% vs 60%,2 mo and 4 mo after surgery,respectively; P =0.009),severity of dysplasia (0% vs 71.4% and 20% vs 85.7%high-grade dysplasia,2 mo and 4 mo after surgery,respectively; P =0.002),and adenocarcinoma incidence (0% vs 57.1% and 0% vs 60%,2 mo and 4 mo after surgery,respectively; P < 0.0001).Treatment with the selective COX-2

  5. Inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase by GSK5750 correlates with slow enzyme-inhibitor dissociation. (United States)

    Beilhartz, Greg L; Ngure, Marianne; Johns, Brian A; DeAnda, Felix; Gerondelis, Peter; Götte, Matthias


    Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

  6. Synthesis, inhibition and binding of simple non-nitrogen inhibitors of monoamine transporters. (United States)

    Petersen, Mikkel Due; Boye, Søren Valdgård; Nielsen, Erik Holm; Willumsen, Jeanette; Sinning, Steffen; Wiborg, Ove; Bols, Mikael


    A series of simple truncated analogues of phenyl tropanes, 2-arylcycloalk-1-enyl carboxylic acid methylesters, were prepared and investigated for their activity towards the dopamine, serotonin and norepinephrine transporters. The compounds were prepared from cyclic ketoesters, which were converted to enolic triflates and reacted with arylboronates using the Suzuki coupling. For comparison the corresponding piperidines were also made and investigated. The new compounds inhibit monoamine-transporters with Ki values ranging from 0.1 to 1000 microM.

  7. Density functional theory and molecular dynamics simulation study on corrosion inhibition performance of mild steel by mercapto-quinoline Schiff base corrosion inhibitor (United States)

    Saha, Sourav Kr.; Ghosh, Pritam; Hens, Abhiram; Murmu, Naresh Chandra; Banerjee, Priyabrata


    Corrosion inhibition mechanism of two mercapto-quinoline Schiff bases, eg., 3-((phenylimino)methyl)quinoline-2-thiol (PMQ) and 3-((5-methylthiazol-2-ylimino)methyl) quinoline-2-thiol (MMQT) on mild steel surface is investigated by quantum chemical calculation and molecular dynamics simulation. Quantum chemical parameters such as EHOMO, ELUMO, energy gap (ΔE), dipolemoment (μ), electronegativity (χ), global hardness (η) and fraction of electron transfers from the inhibitor molecule to the metallic atom surface (ΔN) have been studied to investigate their relative corrosion inhibition performance. Parameters like local reactive sites of the present molecule have been analyzed through Fukui indices. Moreover, adsorption behavior of the inhibitor molecules on Fe (1 1 0) surface have been analyzed using molecular dynamics simulation. The binding strength of the concerned inhibitor molecules on mild steel surface follows the order MMQT>PMQ, which is in good agreement with the experimentally determined inhibition efficiencies. In view of the above, our approach will be helpful for quick prediction of a potential inhibitor from a lot of similar inhibitors and subsequently in their rational designed synthesis for corrosion inhibition application following a wet chemical synthetic route.

  8. Peptide Inhibitor of Complement C1 (PIC1 Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

    Directory of Open Access Journals (Sweden)

    Julia A Sharp

    Full Text Available The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1. In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.

  9. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)


    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  10. HIV protease inhibitors disrupt lipid metabolism by activating endoplasmic reticulum stress and inhibiting autophagy activity in adipocytes.

    Directory of Open Access Journals (Sweden)

    Beth S Zha

    Full Text Available BACKGROUND: HIV protease inhibitors (PI are core components of Highly Active Antiretroviral Therapy (HAART, the most effective treatment for HIV infection currently available. However, HIV PIs have now been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease and metabolic syndrome. Our previous studies have shown that HIV PIs activate endoplasmic reticulum (ER stress and disrupt lipid metabolism in hepatocytes and macrophages. Yet, little is known on how HIV PIs disrupt lipid metabolism in adipocytes, a major cell type involved in the pathogenesis of metabolic syndrome. METHODOLOGY AND PRINCIPAL FINDINGS: Cultured and primary mouse adipocytes and human adipocytes were used to examine the effect of frequently used HIV PIs in the clinic, lopinavir/ritonavir, on adipocyte differentiation and further identify the underlying molecular mechanism of HIV PI-induced dysregulation of lipid metabolism in adipocytes. The results indicated that lopinavir alone or in combination with ritonavir, significantly activated the ER stress response, inhibited cell differentiation, and induced cell apoptosis in adipocytes. In addition, HIV PI-induced ER stress was closely linked to inhibition of autophagy activity. We also identified through the use of primary adipocytes of CHOP(-/- mice that CHOP, the major transcriptional factor of the ER stress signaling pathway, is involved in lopinavir/ritonavir-induced inhibition of cell differentiation in adipocytes. In addition, lopinavir/ritonavir-induced ER stress appears to be associated with inhibition of autophagy activity in adipocytes. CONCLUSION AND SIGNIFICANCE: Activation of ER stress and impairment of autophagy activity are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV patients.

  11. [Arginase inhibitor nor-NOHA induces apoptosis and inhibits invasion and migration of HepG2 cells]. (United States)

    Li, Xiangnan; Zhu, Fangyu; He, Yongsong; Luo, Fang


    Objective To investigate the cell inhibitory effect of arginase inhibitor nor-NOHA on HepG2 hepatocellular carcinoma cells and related mechanism. Methods CCK-8 assay was used to detect the cell proliferation and flow cytometry to detect the apoptosis of HepG2 cells treated with (0, 0.5, 1.0, 2.0, 3.0) ng/μL nor-NOHA. The protein levels of arginase 1 (Arg1), P53, matrix metalloproteinase-2 (MMP-2), E-cadherin (ECD) were determined by Western blotting. Real time quantitative PCR was employed to examine the changes in the mRNA level of inducible nitric oxide synthase (iNOS). Griess assay was used to measure the concentration of nitric oxide (NO) in HepG2 cells. Transwell(TM) assay and wound-healing assay were performed to evaluate the changes of the cell invasion and migration ability, respectively. Results nor-NOHA inhibited the proliferation and induced the apoptosis of HepG2 cells. It also decreased the expression levels of Arg1 and MMP-2, increased the expression levels of P53 and ECD as well as the production of NO; in addition, nor-NOHA inhibited the invasion and migration of HepG2 cells. Conclusion Nor-NOHA can induce cell apoptosis and inhibit the ability of invasion and migration of HepG2 cells by inhibiting Arg1, which is related with the increase of iNOS expression and the high concentration of NO.

  12. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

    Indian Academy of Sciences (India)



    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv)channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertralinedecreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 μM and a slope value (Hillcoefficient) of 0.61. Although the application of 1 μM sertraline did not affect the steady-state activation curves,sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine,had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From theseresults, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptakeinhibition by shifting inactivation curves to a more negative potential.

  13. Colchicine Inhibited the Expression of Tissue Inhibitor of Metalloprotenase-1 and Interleukin-6 in Cultured Activated Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    LI Zesong; CAI Shaoxi; JIANG Yuan; GUO RuiJun; ZHANG Wen


    Cultured HSCs were treated colchicine with different concentrations for 12 h, respectively. The effects of colchicine on HSCs growth were measured by MTT assay. Effects of colchicine on gene expression of HSCs were analysed by using a self-made oligonucleotide microarray. Colchicine inhibited HSCs growth in a dose-dependent manner. After 12 h of treatment with 6.25 mg/L of colchicine, the expression of tissue inhibitor of metalloprotenase1 (TIMP-1) and the expression of interleukin-6 (IL-6) in HSCs were downregulated by 2.3 folds and 2.1 folds, respectively. These results suggest that colchicine's beneficial effects may, at least in part, owe to the inhibitory to the proliferation of HSCs and down-regulation of the expression of both TIMP1 and IL-6 in HSCs.

  14. 一种新型四元阻垢剂的合成和性能研究%Synthesis and Inhibition Efficiency of a Novel Quadripolymer Inhibitor

    Institute of Scientific and Technical Information of China (English)

    张云霞; 吴季怀; 郝三存; 刘明华


    A novel quadripolymer scale inhibitor poly-maleic anhydride-acrylic acid-acrylamide-sodium methallyl sulfonate (PMAAS) was synthesized by solution polymerization with maleic anhydride (MA), acrylic acid (AA),acrylamide (AM), sodium methallyl sulfonate (SMAS), etc. IR spectrum shows that PMAAS contains carbonyl,hydroxyl, phosphatic and sulfonic acid group. SEM indicates that PMAAS blocks the normal growth of scale CaCO3 and CaSO4 crystals. The influences of PMAAS concentration, Ca2+ concentration, temperature and pH value of the system on the inhibition efficiency are investigated. The inhibition efficiency of PMAAS is superior to commercial inhibitors T-225 and XF-192.

  15. NT113, a pan-ERBB inhibitor with high brain penetrance, inhibits the growth of glioblastoma xenografts with EGFR amplification. (United States)

    Yoshida, Yasuyuki; Ozawa, Tomoko; Yao, Tsun-Wen; Shen, Wang; Brown, Dennis; Parsa, Andrew T; Raizer, Jeffrey J; Cheng, Shi-Yuan; Stegh, Alexander H; Mazar, Andrew P; Giles, Francis J; Sarkaria, Jann N; Butowski, Nicholas; Nicolaides, Theodore; James, C David


    This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM.

  16. Histone deacetylase inhibitor upregulates peroxisomal fatty acid oxidation and inhibits apoptotic cell death in abcd1-deficient glial cells.

    Directory of Open Access Journals (Sweden)

    Jaspreet Singh

    Full Text Available In X-ALD, mutation/deletion of ALD gene (ABCD1 and the resultant very long chain fatty acid (VLCFA derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD. The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs (1 and 3 in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL and cell survival (phospho-Erk1/2 proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC inhibitor suberoylanilide hydroxamic acid (SAHA corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

  17. Inhibition of envelope-mediated CD4+-T-cell depletion by human immunodeficiency virus attachment inhibitors. (United States)

    Alexander, Louis; Zhang, Sharon; McAuliffe, Brian; Connors, David; Zhou, Nannon; Wang, Tao; Agler, Michele; Kadow, John; Lin, Pin-Fang


    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) binding induces proapoptotic signals in CD4(+) T cells without a requirement of infection. Defective virus particles, which represent the majority of HIV-1, usually contain a functional Env and therefore represent a potentially significant cause of such CD4(+)-T-cell loss. We reasoned that an HIV-1 inhibitor that prohibits Env-host cell interactions could block the destructive effects of defective particles. HIV-1 attachment inhibitors (AIs), which potently inhibit Env-CD4 binding and subsequent downstream effects of Env, display low-nanomolar antiapoptotic potency and prevent CD4(+)-T-cell depletion from mixed lymphocyte cultures, also with low-nanomolar potency. Specific Env amino acid changes that confer resistance to AI antientry activity eliminate AI antiapoptotic effects. We observed that CD4(+)-T-cell destruction is specific for CXCR4-utilizing HIV-1 strains and that the fusion blocker enfuvirtide inhibits Env-mediated CD4(+)-T-cell killing but is substantially less potent than AIs. These observations, in conjunction with observed antiapoptotic activities of soluble CD4 and the CXCR4 blocker AMD3100, suggest that this AI activity functions through a mechanism common to AI antientry activity, e.g., prevention of Env conformation changes necessary for specific interactions with cellular factors that facilitate viral entry. Our study suggests that AIs, in addition to having potent antientry activity, could contribute to immune system homeostasis in individuals infected with HIV-1 that can engage CXCR4, thereby mitigating the increased risk of adverse clinical events observed in such individuals on current antiretroviral regimens.

  18. Topoisomerase I inhibitors, shikonin and topotecan, inhibit growth and induce apoptosis of glioma cells and glioma stem cells.

    Directory of Open Access Journals (Sweden)

    Feng-Lei Zhang

    Full Text Available Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

  19. Inhibition of RPE cell sterile inflammatory responses and endotoxin-induced uveitis by a cell-impermeable HSP90 inhibitor. (United States)

    Qin, Suofu; Ni, Ming; Wang, Xiuyun; Maurier-Mahé, Florence; Shurland, Dixie-Lee; Rodrigues, Gerard A


    Dying cells release pro-inflammatory molecules, functioning as cytokines to trigger cell/tissue inflammation that is relevant to disease pathology. Heat-shock protein 90 (HSP90) is believed to act as a danger signal for tissue damage once released extracellularly. Potential roles of HSP90 were explored in retinal pigment epithelial (RPE) inflammatory responses to necrosis. Cellular extracts can trigger ARPE-19 cell inflammatory responses, producing cytokines that lead to an increase in ARPE-19 cell monolayer permeability. Addition of recombinant HSP90β mimics the induction of chemokines IL-8 and MCP-1 in cultured RPE cells, suggesting that released HSP90 can incite RPE cell sterile inflammatory responses. Consistent with this, classical HSP90 inhibitors were shown to substantially reduce necrosis-induced cytokine production and permeability increases in ARPE-19 cells. Moreover, a cell-impermeable inhibitor, 17-N,N-dimethylaminoethylamino-17-demethoxy-geldanamycin-N-oxide, also efficiently inhibited necrosis-induced cytokine production and TNF-α/IL-1β-induced increase in ARPE-19 cell permeability in vitro and endotoxin-induced development of uveitis in vivo, suggesting that HSP90 can contribute to necrosis-induced RPE inflammatory responses. Collectively, our data identify HSP90 as a pro-inflammatory molecule in RPE cell sterile inflammatory responses. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State

    Directory of Open Access Journals (Sweden)

    Daniel M. Jones


    Full Text Available One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

  1. PI3KC2{alpha}, a class II PI3K, is required for dynamin-independent internalization pathways

    DEFF Research Database (Denmark)

    Krag, Claudia; Malmberg, Emily Kim; Salcini, Anna Elisabetta


    Increasing evidence indicates that cellular uptake of several molecules can occur independently of functional dynamin, but the molecular players that regulate dynamin-independent endocytosis and the subsequent trafficking steps are still largely unknown. A survival-based short-hairpin (sh) RNA...... screen using a cell line expressing a diphtheria toxin receptor (DTR, officially known as HBEGF) anchored to GPI (DTR-GPI), which internalizes diphtheria toxin (DT, officially known as DTX) in a dynamin-independent manner, identified PI3KC2a, a class II phosphoinositide 3-kinase (PI3K), as a specific...... regulator of dynamin-independent DT internalization. We found that the internalization of several proteins that enter the cell through dynamin-independent pathways led to a relocalization of PI3KC2a to cargo-positive vesicles. Furthermore, downregulation of PI3KC2a impaired internalization of CD59 as well...

  2. Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

    Directory of Open Access Journals (Sweden)

    Anna Sandebring

    Full Text Available PTEN-induced novel kinase 1 (PINK1 mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1 exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

  3. Questioning the reliability of estimates of enzyme inhibitor constant: Case of competitive inhibition

    CERN Document Server

    Dhatt, Sharmistha


    Reliability of kinetic parameters are crucial in understanding enzyme kinetics within cellular system. The present study suggests a few cautions that need introspection for estimation of parameters like K(M), V(max) and K(I) using Lineweaver-Burk plots. The quality of IC(50) too needs a thorough reinvestigation because of its direct link with K(I) and K(M) values. Inhibition kinetics under both steady-state and non-steady-state conditions are studied and errors in estimated parameters are compared against actual values to settle the question of their adequacy.

  4. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  5. Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study

    Directory of Open Access Journals (Sweden)

    Kaile Zhang


    Full Text Available Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001 delivering collagen/poly(l-lactide-co-caprolactone (P(LLA-CL scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM expression in vitro and in vivo. Methods: ICG-001 (1 mg (2 mM was loaded into a (P(LLA-CL scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron and high-performance liquid chromatography (HPLC. Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl-2,5-Diphenyltetrazolium Bromide (MTT assay and scanning electron microscopy (SEM were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-β1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. Results: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more

  6. Revealing the Mechanistic Pathway of Acid Activation of Proton Pump Inhibitors To Inhibit the Gastric Proton Pump: A DFT Study. (United States)

    Jana, Kalyanashis; Bandyopadhyay, Tusar; Ganguly, Bishwajit


    Acid-related gastric diseases are associated with disorder of digestive tract acidification due to the acid secretion by gastric proton pump, H(+),K(+)-ATPase. Omeprazole is one of the persuasive irreversible inhibitor of the proton pump H(+),K(+)-ATPase. However, the reports on the mechanistic pathway of irreversible proton pump inhibitors (PPIs) on the acid activation and formation of disulfide complex are scarce in the literature. We have examined the acid activation PPIs, i.e., timoprazole, S-omeprazole and R-omeprazole using M062X/6-31++G(d,p) in aqueous phase with SMD solvation model. The proton pump inhibitor is a prodrug and activated in the acidic canaliculi of the gastric pump H(+),K(+)-ATPase to sulfenic acid which can either form another acid activate intermediate sulfenamide or a disulfide complex with cysteine amino acid of H(+),K(+)-ATPase. The quantum chemical calculations suggest that the transition state (TS5) for the disulfide complex formation is the rate-determining step of the multistep acid inhibition process by PPIs. The free energy barrier of TS5 is 5.5 kcal/mol higher for timoprazole compared to the S-omeprazole. The stability of the transition state for the formation of disulfide bond between S-omeprazole and cysteine amino acid of H(+),K(+)-ATPase is governed by inter- and intramolecular hydrogen bonding. The disulfide complex for S-omeprazole is thermodynamically more stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole, which corroborates the less efficacy of timoprazole as irreversible PPI for acid inhibition process. It has been speculated that sulfenic acid can either form sulfenamide or a stable disulfide complex with cysteine amino acid residue of H(+),K(+)-ATPase. The M062X/6-31++G(d,p) level of theory calculated results reveal that the formation of tetra cyclic sulfenamide is unfavored by ∼17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation

  7. Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor. (United States)

    Shikata, Yuki; Yoshimaru, Tetsuro; Komatsu, Masato; Katoh, Hiroto; Sato, Reiko; Kanagaki, Shuhei; Okazaki, Yasumasa; Toyokuni, Shinya; Tashiro, Etsu; Ishikawa, Shumpei; Katagiri, Toyomasa; Imoto, Masaya


    Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells. This article is protected by copyright. All rights reserved.

  8. Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn. (United States)

    Korneeva, Vera A; Trubetskov, Mikhail M; Korshunova, Alena V; Lushchekina, Sofya V; Kolyadko, Vladimir N; Sergienko, Olga V; Lunin, Vladimir G; Panteleev, Mikhail A; Ataullakhanov, Fazoil I


    Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.

  9. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang


    Full Text Available Pectinesterase inhibitor (PEI isolated from jelly fig (Ficus awkeotsang Makino is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg. Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B and integrated (Huh7 HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  10. Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas. (United States)

    Naylor, Tara L; Tang, Huaping; Ratsch, Boris A; Enns, Andreas; Loo, Alice; Chen, Liqing; Lenz, Peter; Waters, Nigel J; Schuler, Walter; Dörken, Bernd; Yao, Yung-Mae; Warmuth, Markus; Lenz, Georg; Stegmeier, Frank


    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.

  11. Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors. (United States)

    Flores-Sanchez, Isvett Josefina; Gang, David Roger


    Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively.

  12. Inhibitor ranking through QM based chelation calculations for virtual screening of HIV-1 RNase H inhibition.

    Directory of Open Access Journals (Sweden)

    Vasanthanathan Poongavanam

    Full Text Available Quantum mechanical (QM calculations have been used to predict the binding affinity of a set of ligands towards HIV-1 RT associated RNase H (RNH. The QM based chelation calculations show improved binding affinity prediction for the inhibitors compared to using an empirical scoring function. Furthermore, full protein fragment molecular orbital (FMO calculations were conducted and subsequently analysed for individual residue stabilization/destabilization energy contributions to the overall binding affinity in order to better understand the true and false predictions. After a successful assessment of the methods based on the use of a training set of molecules, QM based chelation calculations were used as filter in virtual screening of compounds in the ZINC database. By this, we find, compared to regular docking, QM based chelation calculations to significantly reduce the large number of false positives. Thus, the computational models tested in this study could be useful as high throughput filters for searching HIV-1 RNase H active-site molecules in the virtual screening process.

  13. Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

    Directory of Open Access Journals (Sweden)

    Wang Lin


    Full Text Available Abstract Background The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin on human retinal pigment epithelial cells is investigated. Methods MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. Results 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. Conclusions 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.

  14. Multiple ascending dose study with the new renin inhibitor VTP-27999: nephrocentric consequences of too much renin inhibition. (United States)

    Balcarek, Joanna; Sevá Pessôa, Bruno; Bryson, Catherine; Azizi, Michel; Ménard, Joël; Garrelds, Ingrid M; McGeehan, Gerard; Reeves, Richard A; Griffith, Sue G; Danser, A H Jan; Gregg, Richard


    This study compared the pharmacodynamic/pharmacokinetic profile of the new renin inhibitor VTP-27999 in salt-depleted healthy volunteers, administered once daily (75, 150, 300, and 600 mg) for 10 days, versus placebo and 300 mg aliskiren. VTP-27999 was well tolerated with no significant safety issues. It was rapidly absorbed, attaining maximum plasma concentrations at 1 to 4 hours after dosing, with a terminal half-life of 24 to 30 hours. Plasma renin activity remained suppressed during the 24-hour dosing interval at all doses. VTP-27999 administration resulted in a dose-dependent induction of renin, increasing the concentration of plasma renin maximally 350-fold. This induction was greater than with aliskiren, indicating greater intrarenal renin inhibition. VTP-27999 decreased plasma angiotensin II and aldosterone. At 24 hours and later time points after dosing on day 10 in the 600-mg group, angiotensin II and aldosterone levels were increased, and plasma renin activity was also increased at 48 and 72 hours, compared with baseline. VTP-27999 decreased urinary aldosterone excretion versus placebo on day 1. On day 10, urinary aldosterone excretion was higher in the 300- and 600-mg VTP-27999 dose groups compared with baseline. VTP-27999 decreased blood pressure to the same degree as aliskiren. In conclusion, excessive intrarenal renin inhibition, obtained at VTP-27999 doses of 300 mg and higher, is accompanied by plasma renin rises, that after stopping drug intake, exceed the capacity of extrarenal VTP-27999 to block fully the enzymatic reaction. This results in significant rises of angiotensin II and aldosterone. Therefore, renin inhibition has an upper limit.

  15. Andrographolide, a Novel NF-κB Inhibitor, Inhibits Vascular Smooth Muscle Cell Proliferation and Cerebral Endothelial Cell Inflammation (United States)

    Chang, Chao-Chien; Duann, Yeh-Fang; Yen, Ting-Lin; Chen, Yu-Ying; Jayakumar, Thanasekaran; Ong, Eng-Thiam; Sheu, Joen-Rong


    Background Aberrant vascular smooth muscle cell (VSMC) proliferation and cerebral endothelial cell (CEC) dysfunction contribute significantly in the pathogenesis of cardiovascular diseases. Therefore, inhibition of these cellular events would be by candidate agents for treating these diseases. In the present study, the mechanism of anti-proliferative and anti-inflammatory effects of andrographolides, a novel nuclear factor-κB inhibitor, was investigated in VSMC and CEC cells. Methods VSMCs and CECs were isolated from rat artery and mouse brain, respectively, and cultured before experimentation. The effect of andro on platelet-derived growth factor-BB (PDGF-BB) induced VSMC cell proliferation was evaluated by cell number, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of extracellular signal regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA), and the effects on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and, cyclooxygenase-2 (COX2) were detected by Western blotting. Results Andro significantly inhibited PDGF-BB (10 ng/ml) induced cell proliferation in a concentration (20-100 μM) dependent manner, which may be due to reducing the expression of ERK1/2, and by inhibiting the expression of PCNA. Andro also remarkably diminished LPS-induced iNOS and COX2 expression. Conclusions The results of this study suggested that the effects of andro against VSMCs proliferation and CECs dysfunction may represent a promising approach for treatment of vascular diseases. PMID:27122804

  16. Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors. (United States)

    Lin, Zeyu; Cantone, Joseph; Lu, Hao; Nowicka-Sans, Beata; Protack, Tricia; Yuan, Tian; Yang, Hong; Liu, Zheng; Drexler, Dieter; Regueiro-Ren, Alicia; Meanwell, Nicholas A; Cockett, Mark; Krystal, Mark; Lataillade, Max; Dicker, Ira B


    HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC50 values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral

  17. Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.

    Directory of Open Access Journals (Sweden)

    Gallen B Triana-Baltzer

    Full Text Available Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase, a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI strain (H5N1. Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

  18. SphK1 inhibitor II (SKI-II) inhibits acute myelogenous leukemia cell growth in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Li; Weng, Wei; Sun, Zhi-Xin; Fu, Xian-Jie; Ma, Jun, E-mail:; Zhuang, Wen-Fang, E-mail:


    Previous studies have identified sphingosine kinase 1 (SphK1) as a potential drug target for treatment of acute myeloid leukemia (AML). In the current study, we investigated the potential anti-leukemic activity of a novel and specific SphK1 inhibitor, SKI-II. We demonstrated that SKI-II inhibited growth and survival of human AML cell lines (HL-60 and U937 cells). SKI-II was more efficient than two known SphK1 inhibitors SK1-I and FTY720 in inhibiting AML cells. Meanwhile, it induced dramatic apoptosis in above AML cells, and the cytotoxicity by SKI-II was almost reversed by the general caspase inhibitor z-VAD-fmk. SKI-II treatment inhibited SphK1 activation, and concomitantly increased level of sphingosine-1-phosphate (S1P) precursor ceramide in AML cells. Conversely, exogenously-added S1P protected against SKI-II-induced cytotoxicity, while cell permeable short-chain ceramide (C6) aggravated SKI-II's lethality against AML cells. Notably, SKI-II induced potent apoptotic death in primary human AML cells, but was generally safe to the human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. In vivo, SKI-II administration suppressed growth of U937 leukemic xenograft tumors in severe combined immunodeficient (SCID) mice. These results suggest that SKI-II might be further investigated as a promising anti-AML agent. - Highlights: • SKI-II inhibits proliferation and survival of primary and transformed AML cells. • SKI-II induces apoptotic death of AML cells, but is safe to normal PBMCs. • SKI-II is more efficient than two known SphK1 inhibitors in inhibiting AML cells. • SKI-II inhibits SphK1 activity, while increasing ceramide production in AML cells. • SKI-II dose-dependently inhibits U937 xenograft growth in SCID mice.

  19. Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition. (United States)

    Micheelsen, Pernille Ollendorff; Vévodová, Jitka; De Maria, Leonardo; Ostergaard, Peter Rahbek; Friis, Esben Peter; Wilson, Keith; Skjøt, Michael


    Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.

  20. Inhibition of in vivo histamine metabolism in rats by foodborne and pharmacologic inhibitors of diamine oxidase, histamine N-methyltransferase, and monoamine oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Hui, J.Y.; Taylor, S.L.


    When (/sup 14/C)histamine was administered orally to rats, an average of 80% of the administered radioactivity was recovered in the urine at the end of 24 hr. About 10% of the total dose was excreted via the feces. Analysis of 4-hr urine samples found imidazoleacetic acid to be the predominant metabolite (60.6%), with N tau-methylimidazoleacetic acid (8.6%), N tau-methylhistamine (7.3%), and N-acetylhistamine (4.5%) to be the minor metabolites. Histamine metabolism was inhibited by simultaneous oral administration of aminoguanidine, isoniazid, quinacrine, cadaverine, putrescine, tyramine, and beta-phenylethylamine. The administration of inhibitors resulted in an increased amount of unmetabolized histamine and a decreased amount of metabolites reaching the urine. Pharmacologic inhibitors were found to be more potent and have a longer duration of action than foodborne ones. The inhibitors could potentiate food poisoning caused by histamine by inhibiting its metabolism.

  1. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    Energy Technology Data Exchange (ETDEWEB)

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)


    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  2. In vitro and ex vivo inhibition of human telomerase by anti-HIV nucleoside reverse transcriptase inhibitors (NRTIs but not by non-NRTIs.

    Directory of Open Access Journals (Sweden)

    Kyle R Hukezalie

    Full Text Available Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs, including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that

  3. Smed-dynA-1 is a planarian nervous system specific dynamin 1 homolog required for normal locomotion

    Directory of Open Access Journals (Sweden)

    Jared A. Talbot


    Full Text Available Dynamins are GTPases that are required for separation of vesicles from the plasma membrane and thus are key regulators of endocytosis in eukaryotic cells. This role for dynamin proteins is especially crucial for the proper function of neurons, where they ensure that synaptic vesicles and their neurotransmitter cargo are recycled in the presynaptic cell. Here we have characterized the dynamin protein family in the freshwater planarian Schmidtea mediterranea and showed that it possesses six dynamins with tissue specific expression profiles. Of these six planarian homologs, two are necessary for normal tissue homeostasis, and the loss of another, Smed-dynA-1, leads to an abnormal behavioral phenotype, which we have quantified using automated center of mass tracking. Smed-dynA-1 is primarily expressed in the planarian nervous system and is a functional homolog of the mammalian Dynamin I. The distinct expression profiles of the six dynamin genes makes planarians an interesting new system to reveal novel dynamin functions, which may be determined by their differential tissue localization. The observed complexity of neurotransmitter regulation combined with the tools of quantitative behavioral assays as a functional readout for neuronal activity, renders planarians an ideal system for studying how the nervous system controls behavior.

  4. Smed-dynA-1 is a planarian nervous system specific dynamin 1 homolog required for normal locomotion. (United States)

    Talbot, Jared A; Currie, Ko W; Pearson, Bret J; Collins, Eva-Maria S


    Dynamins are GTPases that are required for separation of vesicles from the plasma membrane and thus are key regulators of endocytosis in eukaryotic cells. This role for dynamin proteins is especially crucial for the proper function of neurons, where they ensure that synaptic vesicles and their neurotransmitter cargo are recycled in the presynaptic cell. Here we have characterized the dynamin protein family in the freshwater planarian Schmidtea mediterranea and showed that it possesses six dynamins with tissue specific expression profiles. Of these six planarian homologs, two are necessary for normal tissue homeostasis, and the loss of another, Smed-dynA-1, leads to an abnormal behavioral phenotype, which we have quantified using automated center of mass tracking. Smed-dynA-1 is primarily expressed in the planarian nervous system and is a functional homolog of the mammalian Dynamin I. The distinct expression profiles of the six dynamin genes makes planarians an interesting new system to reveal novel dynamin functions, which may be determined by their differential tissue localization. The observed complexity of neurotransmitter regulation combined with the tools of quantitative behavioral assays as a functional readout for neuronal activity, renders planarians an ideal system for studying how the nervous system controls behavior.

  5. Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036 (United States)

    Chan, Wayne W.; Wise, Scott C.; Kaufman, Michael D.; Ahn, Yu Mi; Ensinger, Carol L.; Haack, Torsten; Hood, Molly M.; Jones, Jennifer; Lord, John W.; Lu, Wei Ping; Miller, David; Patt, William C.; Smith, Bryan D.; Petillo, Peter A.; Rutkoski, Thomas J.; Telikepalli, Hanumaiah; Vogeti, Lakshminarayana; Yao, Tony; Chun, Lawrence; Clark, Robin; Evangelista, Peter; Gavrilescu, L. Cristina; Lazarides, Katherine; Zaleskas, Virginia M.; Stewart, Lance J.; Van Etten, Richard A.; Flynn, Daniel L.


    Summary Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for chronic myeloid leukemia (CML) patients. Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead “switch-control” inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1T315I-expressing cell lines, prolongs survival in mouse models of T315I-mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph+ leukemia. PMID:21481795

  6. FGF21 promotes endothelial cell angiogenesis through a dynamin-2 and Rab5 dependent pathway.

    Directory of Open Access Journals (Sweden)

    Usman Yaqoob

    Full Text Available Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling.

  7. FGF21 Promotes Endothelial Cell Angiogenesis through a Dynamin-2 and Rab5 Dependent Pathway (United States)

    Yaqoob, Usman; Jagavelu, Kumaravelu; Shergill, Uday; de Assuncao, Thiago; Cao, Sheng; Shah, Vijay H.


    Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling. PMID:24848261

  8. Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy.

    NARCIS (Netherlands)

    Claeys, K.G.; Zuchner, S.; Kennerson, M.; Berciano, J.; Garcia, A.; Verhoeven, K.; Storey, E.; Merory, J.R.; Bienfait, H.M.; Lammens, M.M.Y.; Nelis, E.; Baets, J.; Vriendt, E. De; Berneman, Z.N.; Veuster, I. De; Vance, J.M.; Nicholson, G.; Timmerman, V.; Jonghe, P. de


    Dominant intermediate Charcot-Marie-Tooth neuropathy type B is caused by mutations in dynamin 2. We studied the clinical, haematological, electrophysiological and sural nerve biopsy findings in 34 patients belonging to six unrelated dominant intermediate Charcot-Marie-Tooth neuropathy type B familie

  9. Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles

    NARCIS (Netherlands)

    van Dam, EM; Stoorvogel, W


    Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the traps-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that

  10. Specific interaction with cardiolipin triggers functional activation of Dynamin-Related Protein 1.

    Directory of Open Access Journals (Sweden)

    Itsasne Bustillo-Zabalbeitia

    Full Text Available Dynamin-Related Protein 1 (Drp1, a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G, bundle signaling element (BSE and stalk domains. However, instead of the lipid-interacting Pleckstrin Homology (PH domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL. Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.

  11. Phenotypic spectrum of dynamin 2 mutations in Charcot-Marie-Tooth neuropathy

    NARCIS (Netherlands)

    K.G. Claeys; S. Züchner; M. Kennerson; J. Berciano; A. Garcia; K. Verhoeven; E. Storey; J.R. Merory; H.M.E. Bienfait; M. Lammens; E. Nelis; J. Baets; E. de Vriendt; Z.N. Berneman; I. de Veuster; J.M. Vance; G. Nicholson; V. Timmerman; P. de Jonghe


    Dominant intermediate Charcot-Marie-Tooth neuropathy type B is caused by mutations in dynamin 2. We studied the clinical, haematological, electrophysiological and sural nerve biopsy findings in 34 patients belonging to six unrelated dominant intermediate Charcot-Marie-Tooth neuropathy type B familie

  12. LeoA, B and C from enterotoxigenic Escherichia coli (ETEC are bacterial dynamins.

    Directory of Open Access Journals (Sweden)

    Katharine A Michie

    Full Text Available Escherichia coli (ETEC strain H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP. Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.

  13. Sustained Exocytosis after Action Potential-Like Stimulation at Low Frequencies in Mouse Chromaffin Cells Depends on a Dynamin-Dependent Fast Endocytotic Process (United States)

    Moya-Díaz, José; Álvarez, Yanina D.; Montenegro, Mauricio; Bayonés, Lucas; Belingheri, Ana V.; González-Jamett, Arlek M.; Cárdenas, Ana M.; Marengo, Fernando D.


    Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 ± 0.11 s, fast enough to maintain synchronous exocytosis at 0.2–0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 ± 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies. PMID:27507935


    Directory of Open Access Journals (Sweden)

    José Moya-Díaz


    Full Text Available Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls. The exocytosis triggered by APls (ETAP represents a fraction (40% of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 +/- 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ=0.53±0.01 s. In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz. Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies.

  15. Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline

    Directory of Open Access Journals (Sweden)

    Ruffieux Jean


    Full Text Available Abstract Background Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells. Methods Human endothelial cells were isolated from umbilical veins and stimulated with TNFα (10 ng/ml for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1, intercellular adhesion molecule-1 (ICAM-1, and E-selectin were assessed by immunoblotting. Results The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFα was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl-L-cysteine (BEC had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFα-induced activation of NF-κB, JNK, p38mapk, while it inhibited p70s6k (S6K1 activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFα. Conclusion The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

  16. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O


    of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing....

  17. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available BACKGROUND: HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages. METHODOLOGY AND PRINCIPAL FINDINGS: Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp. CONCLUSION AND SIGNIFICANCE: HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  18. The endoplasmic reticulum stress inhibitor salubrinal inhibits the activation of autophagy and neuroprotection induced by brain ischemic preconditioning

    Institute of Scientific and Technical Information of China (English)

    Bo GAO; Xiang-yang ZHANG; Rong HAN; Tong-tong ZHANG; Cheng CHEN; Zheng-hong QIN; Rui SHENG


    Aim:To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.Methods:The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC,and was occluded permanently 24 h later to induce permanent focal ischemia (PFI).ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC.Infarct volume and motor behavior deficits were examined after the ischemic insult.The protein levels of LC3,p62,HSP70,glucose-regulated protein 78 (GRP 78),p-elF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting.LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.Results:Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC,as evidenced by the significant increases in mortality,infarct volume and motor deficits after PFI.At the molecular levels,pretreatment with SAL (150 pmol) significantly increased p-elF2α level,and decreased GRP78 level after PFI,suggesting that SAL effectively inhibited ER stress in the cortex.Furthermore,the pretreatment with SAL blocked the IPC-induced upregulation of LC3-Ⅱ and downregulation of p62 in the cortex,thus inhibiting the activation of autophagy.Moreover,SAL blocked the upregulation of HSP70,but significantly increased the cleaved caspase-12 level,thus promoting ER stress-dependent apoptotic signaling in the cortex.Conclusion:ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.

  19. L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Hongbiao Huang

    Full Text Available L-carnitine (LC is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1 LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2 LC treatment selectively induces the expression of p21(cip1 gene, mRNA and protein in cancer cells but not p27(kip1; (4 LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5 LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6 LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1 gene but not p27(kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.

  20. Enzyme inhibition studies by integrated Michaelis-Menten equation considering simultaneous presence of two inhibitors when one of them is a reaction product. (United States)

    Bezerra, Rui M F; Pinto, Paula A; Fraga, Irene; Dias, Albino A


    To determine initial velocities of enzyme catalyzed reactions without theoretical errors it is necessary to consider the use of the integrated Michaelis-Menten equation. When the reaction product is an inhibitor, this approach is particularly important. Nevertheless, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction product. The occurrence of these situations emphasizes the importance of extending the integrated Michaelis-Menten equation, assuming the simultaneous presence of more than one inhibitor because reaction product is always present. This methodology is illustrated with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction product, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination of the kinetic models was carried out based on Akaike information criterion. This work presents a methodology that can be used to develop an automated process, to discriminate in real time the inhibition type and kinetic constants as data (product vs. time) are achieved by the spectrophotometer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies. (United States)

    Prasad, G Shyam; Srisailam, K; Sashidhar, R B


    Specific inhibitors of Cytochrome P4502C9 enzyme (CYP2C9) viz. clopidogrel, fenofibrate fluvoxamine and sertraline at concentration of 50, 100, 150 and 200 µM were employed to investigate the nature of enzyme involved in bioconversion of meloxicam to its main metabolite 5-OH methyl meloxicam by Cunninghamella blakesleeana. Virtual screening for interaction of specific CYP2C9 inhibitors with human CYP2C9 enzyme was performed by molecular docking using Auto dock vina 4.2 version. The in silico studies were further substantiated by in vitro studies, which indicated fenofibrate to be a potent inhibitor of CYP2C9 enzyme followed by sertraline, clopidogrel and fluvoxamine, respectively. Two-stage fermentation protocol was followed to study metabolism of meloxicam and its inhibition by different CYP2C9 inhibitors. Meloxicam metabolites were identified using HPLC, LC-MS analysis and based on previous reports, as 5-OH methyl meloxicam (M1), 5-carboxy meloxicam (M2) and an unidentified metabolite (M3). All the inhibitors tested in the study showed a clear concentration dependent inhibition of meloxicam metabolism. The results suggest that the enzymes involved in metabolism of meloxicam in C. blakesleeana are akin to mammalian metabolism. Hence, C. blakesleeana can be used as a model organism in studying drug interactions and also in predicting mammalian drug metabolism.

  2. A glycoprotein α-amylase inhibitor from Withania somnifera differentially inhibits various α-amylases and affects the growth and development of Tribolium castaneum. (United States)

    Kasar, Sainath S; Marathe, Kiran R; Bhide, Amey J; Herwade, Abhijeet P; Giri, Ashok P; Maheshwari, Vijay L; Pawar, Pankaj K


    Identification and characterisation of plant defensive molecules enrich our resources to design crop protection strategies. In particular, plant-derived proteinaceous inhibitor(s) of insect digestive enzymes appear to be a safe, sustainable and attractive option. A glycoprotein having non-competitive α-amylase inhibitory activity with a molecular weight of 8.3 kDa was isolated and purified from seeds of Withania somnifera α-amylase inhibitor (WSAI). Its mass spectrometry analysis revealed 59% sequence coverage with Wrightide II-type α-amylase inhibitor from Wrightia religiosa. A dose-dependent inhibition of α-amylases from Aspergillus oryzae, Bacillus subtilis, Helicoverpa armigera and Tribolium castaneum was recorded. Interestingly, WSAI did not inhibit human salivary α-amylase significantly. When adults of T. castaneum were fed with WSAI (1.6 mg g(-1) ), decrease in consumption, growth and efficiency of conversion of ingested food was evident, along with over fourfold increases in feeding deterrence index. A decline in larval residual α-amylase activity after feeding of WSAI resulted in a reduction in longevity of T. castaneum. The study reflects the significance of WSAI in affecting the overall growth and development of T. castaneum. Pre- and post-harvest pest resistive capability makes WSAI a potential candidate for insect pest management. Further, the effectiveness of this inhibitor could be explored either in formulations or through a transgenic approach. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  3. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases. (United States)

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica


    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. The kinase-inhibitor sorafenib inhibits multiple steps of the Hepatitis C Virus infectious cycle in vitro. (United States)

    Descamps, Véronique; Helle, François; Louandre, Christophe; Martin, Elodie; Brochot, Etienne; Izquierdo, Laure; Fournier, Carole; Hoffmann, Thomas W; Castelain, Sandrine; Duverlie, Gilles; Galmiche, Antoine; François, Catherine


    Hepatitis C Virus (HCV) chronic infection is a major cause of hepatocellular carcinoma. Sorafenib is the only medical treatment that has been approved for the treatment of this cancer. It is a multikinase inhibitor with anti-tumor activity against a wide variety of cancers. Sorafenib blocks angiogenesis and tumor cell proliferation through inhibition of kinases, such as VEGFR2, PDGFR, or the serine/threonine kinases RAF. Previous studies have reported an anti-HCV effect of sorafenib in vitro, but various mechanisms of action have been described. The aim of this study was to clarify the action of sorafenib on the complete HCV infectious cycle. In order to examine the action of sorafenib on all steps of the HCV infectious cycle, we used a combination of validated cell culture models, based on the HuH-7 reference cell line and primary human hepatocytes. We found that sorafenib blocks HCV infection by altering the viral entry step and the production of viral particles. Moreover, we observed that treatment with sorafenib lead to a modification of Claudin-1 expression and localization, which could partly be responsible for the anti-HCV effect. Collectively, our findings confirm the anti-HCV effect of sorafenib in vitro, while highlighting the complexity of the action of sorafenib on the HCV infectious cycle.

  5. BACE1 Dynamics Upon Inhibition with a BACE Inhibitor and Correlation to Downstream Alzheimer's Disease Markers in Elderly Healthy Participants. (United States)

    Timmers, Maarten; Barão, Soraia; Van Broeck, Bianca; Tesseur, Ina; Slemmon, John; De Waepenaert, Katja; Bogert, Jennifer; Shaw, Leslie M; Engelborghs, Sebastiaan; Moechars, Dieder; Mercken, Marc; Van Nueten, Luc; Tritsmans, Luc; de Strooper, Bart; Streffer, Johannes Rolf


    The β-site amyloid-β protein precursor (AβPP) cleaving enzyme-1 (BACE1) is the rate limiting enzyme in the generation of amyloid-β peptide (Aβ) from AβPP, one of the major pathways in Alzheimer's disease (AD) pathology. Increased BACE1 levels and activity have been reported in the brain of patients with sporadic AD. Therefore, changes of BACE1 levels in the cerebrospinal fluid (CSF) have also been investigated as a possible biomarker of the disease. We analyzed BACE1 levels in CSF of elderly healthy participants before and after chronic treatment with a BACE inhibitor (BACEi) and evaluated the correlation between BACE1 levels and downstream AD markers. Overall, BACE1 CSF levels showed strong correlations to all downstream AD markers investigated. This is the first reported finding that shows BACE1 levels in CSF were well correlated to its end product Aβ1 - 42. As previously described, BACE1 levels were strongly correlated to total-tau and phosphorylated tau levels in CSF. Generally, chronic BACE inhibition did not influence BACE1 CSF protein levels. Follow-up studies including early-stage AD pathophysiology and prodromal AD patients will help to understand the importance of measuring BACE1 routinely in daily clinical practice and AD clinical trials.

  6. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)


    The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

  7. Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : Understanding the basis of differential inhibition and the role of water

    Directory of Open Access Journals (Sweden)

    Mukhopadhayay Bishnu P


    Full Text Available Abstract Background This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition. Results The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition. Conclusion The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn...H2O....Sn of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design.

  8. Concerted actions of ameliorated colitis, aberrant crypt foci inhibition and 15-hydroxyprostaglandin dehydrogenase induction by sonic hedgehog inhibitor led to prevention of colitis-associated cancer. (United States)

    Kangwan, Napapan; Kim, Yoon-Jae; Han, Young-Min; Jeong, Migyeong; Park, Jong-Min; Hahm, Ki-Baik


    The sonic hedgehog (Shh) signaling has been known to contribute to carcinogenesis in organ, where hedgehog exerted organogenesis and in cancers, which are developed based on mutagenic inflammation. Therefore, colitis-associated cancer (CAC) can be a good model to prove whether Shh inhibitors can be applied to prevent, as the efforts to discover potent anti-inflammatory agent are active to prevent CAC. Here, under the hypothesis that Shh inhibitors can prevent CAC, mouse model was generated to develop CAC by azoxymethane (AOM)-initiated, dextran sodium sulfate-promoted carcinogenesis. Shh inhibitors, cerulenin and itraconazole were treated by oral gavage and the mice were sacrificed at early phase of 3 weeks and late phase of 16 weeks. Compared to control group, the number of aberrant crypt foci at 3 weeks and tumor incidence at 16 weeks were all significantly decreased with Shh inhibitor. Significant attenuations of macrophage infiltration accompanied with significant decreases of IL-6, COX-2, STAT3 and NF-κB as well as significant ameliorations of β-catenin nuclear translocation, cyclin D1 and CDK4 were imposed with Shh inhibitors. Especially, CAC was accompanied with significant cancellation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), but their levels were significantly preserved with Shh inhibitors. Among inflammatory mediators, significantly decreased levels of IL-6 and TNF-α, regulated with repressed NF-κb and STAT3, were prominent with Shh inhibitor, whereas significant inductions of apoptosis were noted with Shh inhibitors. In conclusion, Shh inhibitors significantly prevented CAC covering either ameliorating oncogenic inflammation or suppressing tumor proliferation, especially supported with significant inhibition of IL-6 and STAT3 signaling, 15-PGDH preservation and apoptosis induction.

  9. Inhibition of mTOR is required for optimal antitumor effect of HER2 inhibitors against HER2-overexpressing cancer cells (United States)

    Miller, Todd W.; Forbes, James T.; Shah, Chirayu; Wyatt, Shelby K.; Manning, H. Charles; Olivares, Maria G.; Sanchez, Violeta; Dugger, Teresa C.; Granja, Nara de Matos; Narasanna, Archana; Cook, Rebecca S.; Kennedy, J. Phillip; Lindsley, Craig W.; Arteaga, Carlos L.


    Purpose A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mTOR is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design Immunocompetent mice bearing HER2-positive mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG-PET. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26/26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phospho-S6 and Ki67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phospho-S6 and growth in TSC2-expressing but not in TSC2-knockdown cells. Conclusions Inhibition of PI3K and mTOR are required for the growth inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. PMID:19934303

  10. Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

    Directory of Open Access Journals (Sweden)

    Alok Pant

    Full Text Available Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473-AKT in a dose-dependent (10 nM to 1 uM and time-dependent manner (0.5 h to 24 h. MK-2206 inhibited levels of p(Thr308-AKT and a downstream target, p(Thr246-PRAS40, but did not change levels of p(Thr202/Tyr204ERK or p(Thr13/Tyr185SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67 and the most apoptosis (cleaved caspase-3 of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone

  11. The CamKKβ Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells. (United States)

    Jurcsisn, Jennifer G; Pye, Richard L; Ali, Jon; Barr, Barbara L; Wyatt, Christopher N


    It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO609 and Ca(2+) imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca(2+) ([Ca(2+)](I)). Furthermore, STO609 (100 μM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK(Ca) inhibitor paxilline.Taken together these data suggest that ST0609 should be used with caution during Ca(2+) imaging studies as it can directly interact with Ca(2+) binding dyes. The rapid inhibitory effect of STO609 on BK(Ca) was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK(Ca), inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BK(Ca). Future work will determine if the inhibition of BK(Ca) is via CamKKβ or via an off target action of STO609 on the channel itself.

  12. 6-Cyclohexylmethyl-3-hydroxypyrimidine-2,4-dione as an inhibitor scaffold of HIV reverase transcriptase: Impacts of the 3-OH on inhibiting RNase H and polymerase. (United States)

    Tang, Jing; Kirby, Karen A; Huber, Andrew D; Casey, Mary C; Ji, Juan; Wilson, Daniel J; Sarafianos, Stefan G; Wang, Zhengqiang


    3-Hydroxypyrimidine-2,4-dione (HPD) represents a versatile chemical core in the design of inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H and integrase strand transfer (INST). We report herein the design, synthesis and biological evaluation of an HPD subtype (4) featuring a cyclohexylmethyl group at the C-6 position. Antiviral testing showed that most analogues of 4 inhibited HIV-1 in the low nanomolar to submicromolar range, without cytotoxicity at concentrations up to 100 μM. Biochemically, these analogues dually inhibited both the polymerase (pol) and the RNase H functions of RT, but not INST. Co-crystal structure of 4a with RT revealed a nonnucleoside RT inhibitor (NNRTI) binding mode. Interestingly, chemotype 11, the synthetic precursor of 4 lacking the 3-OH group, did not inhibit RNase H while potently inhibiting pol. By virtue of the potent antiviral activity and biochemical RNase H inhibition, HPD subtype 4 could provide a viable platform for eventually achieving potent and selective RNase H inhibition through further medicinal chemistry. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption.

    Directory of Open Access Journals (Sweden)

    An Qin

    Full Text Available Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.

  14. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways. (United States)

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D


    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

  15. Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2). (United States)

    Xu, Xiaoping; Mikhailova, Margarita; Chen, Zhihua; Pal, Sanjay; Robichaud, Trista K; Lafer, Eileen M; Baber, Sam; Steffensen, Bjorn


    Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2(E404A) and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K(d) of 3.9×10(-7)M and 1.7×10(-7)M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2(E404A) and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.

  16. Atorvastatin increases dynamin 1 expression in hippocampal CA1 region in a rat model of vascular dementia

    Institute of Scientific and Technical Information of China (English)

    Qinghua Li; Wensheng Zhou


    The current study examined a rat model of vascular dementia. The model rats exhibited obvious morphological and ultrastructural changes in neurons in the brain, and significantly reduced dynamin 1 expression in hippocampal CA1 region along with decreased learning and memory performance. Following atorvastatin treatment, the morphology and ultrastructure of cells in the model rat brain were significantly improved, dynamin 1 expression in hippocampal CA1 region was significantly enhanced, and learning and memory ability was significantly improved. The results demonstrated that impaired learning and memory abilities in vascular dementia model rats were closely correlated with decreased dynamin 1 expression. These findings indicate that atorvastatin can protect model rats against cognitive impairment by increasing dynamin 1 expression.

  17. Crystal structure of the GTPase domain and the bundle signalling element of dynamin in the GDP state. (United States)

    Anand, Roopsee; Eschenburg, Susanne; Reubold, Thomas F


    Dynamin is the prototype of a family of large multi-domain GTPases. The 100 kDa protein is a key player in clathrin-mediated endocytosis, where it cleaves off vesicles from membranes using the energy from GTP hydrolysis. We have solved the high resolution crystal structure of a fusion protein of the GTPase domain and the bundle signalling element (BSE) of dynamin 1 liganded with GDP. The structure provides a hitherto missing snapshot of the GDP state of the hydrolytic cycle of dynamin and reveals how the switch I region moves away from the active site after GTP hydrolysis and release of inorganic phosphate. Comparing our structure of the GDP state with the known structures of the GTP state, the transition state and the nucleotide-free state of dynamin 1 we describe the structural changes through the hydrolytic cycle.

  18. Release Profile and Inhibition Test of The Nanoparticles A. Paniculata Extract as Inhibitor of α-Glucosidase in The Process of Carbohydrates Breakdown Into Glucose Diabetes Mellitus (United States)

    Imansari, Farisa; Sahlan, Muhammad; Arbianti, Rita


    Andrographis paniculata (A.paniculata) contain the main active substances Andrographolide which helps lower glucose levels in diabetics by inhibiting the enzyme α-glucosidase. The ability of the extract A.paniculata in lowering glucose levels will increase with the technique encapsulation with a coating of composition Chitosan-STPP as a drug delivery to the target organ. This study aimed to get an overview of A.paniculata release profile of nanoparticles in a synthetic fluid media with various concentrations of coating and inhibition testing nasty shard extract in inhibiting the enzyme α-glucosidase. This research resulted in nanoparticles by coating efficiency and loading capacity of chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. chitosan greatest variation of 2% and 1% STPP 60% and 46.29%. The ability of A.paniculata extracts as α-glucosidase enzyme inhibitors has been demonstrated in this study, the percent inhibition of 33.17%.

  19. Real time enzyme inhibition assays provide insights into differences in binding of neuraminidase inhibitors to wild type and mutant influenza viruses.

    Directory of Open Access Journals (Sweden)

    Susan Barrett

    Full Text Available The influenza neuraminidase (NA inhibitors zanamivir, oseltamivir and peramivir were all designed based on the knowledge that the transition state analogue of the cleaved sialic acid, 2-deoxy,2,3-dehydro N-acetyl neuraminic acid (DANA was a weak inhibitor of NA. While DANA bound rapidly to the NA, modifications leading to the improved potency of these new inhibitors also conferred a time dependent or slow binding phenotype. Many mutations in the NA leading to decreased susceptibility result in loss of slow binding, hence this is a phenotypic marker of many but not all resistant NAs. We present here a simplified approach to determine whether an inhibitor is fast or slow binding by extending the endpoint fluorescent enzyme inhibition assay to a real time assay and monitoring the changes in IC(50s with time. We carried out two reactions, one with a 30 min preincubation with inhibitor and the second without. The enzymatic reaction was started via addition of substrate and IC(50s were calculated after each 10 min interval up to 60 min. Results showed that without preincubation IC(50s for the wild type viruses started high and although they decreased continuously over the 60 min reaction time the final IC(50s remained higher than for pre-incubated samples. These results indicate a slow equilibrium of association and dissociation and are consistent with slow binding of the inhibitors. In contrast, for viruses with decreased susceptibility, preincubation had minimal effect on the IC(50s, consistent with fast binding. Therefore this modified assay provides additional phenotypic information about the rate of inhibitor binding in addition to the IC(50, and critically demonstrates the differential effect of incubation times on the IC(50 and K(i values of wild type and mutant viruses for each of the inhibitors.

  20. Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events. (United States)

    Sreedhar, Amere Subbarao; Mihály, Katalin; Pató, Bálint; Schnaider, Tamás; Steták, Attila; Kis-Petik, Katalin; Fidy, Judit; Simonics, Tibor; Maraz, Anna; Csermely, Péter


    The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.

  1. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells (United States)

    Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li


    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  2. Ability of Bruton's Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling. (United States)

    Bender, Andrew T; Gardberg, Anna; Pereira, Albertina; Johnson, Theresa; Wu, Yin; Grenningloh, Roland; Head, Jared; Morandi, Federica; Haselmayer, Philipp; Liu-Bujalski, Lesley


    Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. KN-93, a specific inhibitor of CaMK Ⅱ inhibits human hepatic stellate cell proliferation in vitro

    Institute of Scientific and Technical Information of China (English)

    Ping An; Jun-Yong Zhu; Yan Yang; Peng Lv; Yi-Hao Tian; Ming-Kai Chen; He-Sheng Luo


    AIM: To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate ceils.METHODS: Human hepatic stellate cells (LX-2) were incubated with various concentrations (0-50 μmol/L) of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting.RESULTS: KN-93 (5-50 μmol/L) decreased the proliferation of human hepatic stellate cells in a dosedependent manner from 81.76% (81.76% + 2.58% vs 96.63% + 2.69%, P < 0.05) to 27.15% (27.15% + 2.86% vs 96.59% + 2.44%, P < 0.01) after 24 h treatment.Incubation of 10 μmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21.CONCLUSION: KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.

  4. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  5. MALT1 inhibitors prevent the development of DSS-induced experimental colitis in mice via inhibiting NF-κB and NLRP3 inflammasome activation. (United States)

    Liu, Wen; Guo, Wenjie; Hang, Nan; Yang, Yuanyuan; Wu, Xuefeng; Shen, Yan; Cao, Jingsong; Sun, Yang; Xu, Qiang


    Mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1), a paracaspase and essential regulator for nuclear factor kB (NF-κB) activation, plays an important role in innate and adaptive immunity. Suppression of MALT1 protease activity with small molecule inhibitors showed promising efficacies in subtypes of B cell lymphoma and improvement in experimental autoimmune encephalomyelitis model. However, whether MALT1 inhibitors could ameliorate colitis remains unclear. In the present study, we examined the pharmacological effect of two specific MALT1 inhibitors MI-2 and mepazine on the dextran sulfate sodium (DSS)-induced experimental colitis in mice, followed by mechanistic analysis on NF-κB and NLRP3 inflammasome activation. Treatment with MI-2 and mepazine dose-dependently attenuated symptoms of colitis in mice, evidenced by reduction in the elevated disease activity index, the shortening of colon length as well as the histopathologic improvement. Moreover, protein and mRNA levels of DSS-induced proinflammatory cytokines in colon, including TNF, IL-1β, IL-6, IL-18, IL-17A and IFN-γ, were markedly suppressed by MALT1 inhibitors. The underlying mechanisms for the protective effect of MALT1 inhibitors in DSS-induced colitis may be attributed to its inhibition on NF-κB and NLRP3 inflammasome activation in macrophages. The in vitro study showed that MALT1 inhibitors decreased production of IL-1β/IL-18 in phorbol myristate acetate-differentiated THP-1 cells and bone marrow derived macrophage via suppressing the activation of NF-κB and NLRP3 inflammasome. Taken together, our results demonstrated that inhibition of the protease activity of MALT1 might be a viable strategy to treat inflammatory bowel disease and the NLRP3 inflammasome and NF-κB activation are critical components in MALT1 signaling cascades in this disease model.

  6. Cystic fibrosis transmembrane regulator inhibitors CFTR(inh)-172 and GlyH-101 target mitochondrial functions, independently of chloride channel inhibition. (United States)

    Kelly, Mairead; Trudel, Stephanie; Brouillard, Franck; Bouillaud, Frederick; Colas, Julien; Nguyen-Khoa, Thao; Ollero, Mario; Edelman, Aleksander; Fritsch, Janine


    Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR(inh)-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR(inh)-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR(inh)-172, but not GlyH-101, induced nuclear translocation of nuclear factor-kappaB (NF-kappaB). CFTR(inh)-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-kappaB signaling pathway, independently of CFTR inhibition.

  7. Inhibition by a selective IkappaB kinase-2 inhibitor of interleukin-1-induced collagen degradation by corneal fibroblasts in three-dimensional culture. (United States)

    Kondo, Yukiko; Fukuda, Ken; Adachi, Tadafumi; Nishida, Teruo


    Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-kappaB and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-kappaB activation is mediated by phosphorylation of the inhibitor IkappaB by IkappaB kinase (IKK)-2 and consequent IkappaB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IkappaBalpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. IL-1beta-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration- and time-dependent manner. TPCA-1 inhibited the IL-1beta-induced expression of MMP-1, -3, and -9 in these cells at both the mRNA and protein levels and the IL-1beta-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IkappaBalpha and the nuclear translocation of NF-kappaB induced by IL-1beta. An IKK-2 inhibitor blocked IL-1beta-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-kappaB signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.

  8. TW-37, a Small-Molecule Inhibitor of Bcl-2, Inhibits Cell Growth and Induces Apoptosis in Pancreatic Cancer: Involvement of Notch-1 Signaling Pathway



    Overexpression of Bcl-2 family proteins has been found in a variety of aggressive human carcinomas, including pancreatic cancer, suggesting that specific agents targeting Bcl-2 family proteins would be valuable for pancreatic cancer therapy. We have previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in pancreatic cancer. However, the precise role and the molecular mechanism of action of TW-37 have not been fully elu...

  9. Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and /sup 3/H-mazindol binding in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Angel, I.; Taranger, M.A.; Claustre, Y.; Scatton, B.; Langer, S.Z.


    The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation was obtained between the ED/sub 50/ values of anorectic action and the ED/sub 50/ values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced (/sup 3/H)-mazindol from its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive. Excluding zimelidine, a good correlation was obtained between the affinities of these drugs for (/sup 3/H)-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with (/sup 3/H)-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.

  10. Inhibition of Mcl-1 with the pan–Bcl-2 family inhibitor (–)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia (United States)

    Pan, Rongqing; Ruvolo, Vivian R.; Wei, Jun; Konopleva, Marina; Reed, John C.; Pellecchia, Maurizio; Ruvolo, Peter P.


    Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan–Bcl-2 inhibitor (–)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (–)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan–Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (–)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (–)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan–Bcl-2 inhibition by an Mcl-1–targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment. PMID:26045609

  11. Carbonic anhydrase inhibitors: synthesis and inhibition of the human carbonic anhydrase isoforms I, II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties. (United States)

    Sethi, Kalyan K; Verma, Saurabh M; Tanç, Muhammet; Purper, Gaultier; Calafato, Gaetan; Carta, Fabrizio; Supuran, Claudiu T


    A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1-8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (Kis in the range of 295-10,000 nM), but were more effective hCA II inhibitors (Kis of 1.7-887 nM). The tumor-associated hCA IX was also inhibited, with Kis in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90-3,746 nM. The structure-activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Appropriate risk criteria for OATP inhibition at the drug discovery stage based on the clinical relevancy between OATP inhibitors and drug-induced adverse effect. (United States)

    Nakakariya, Masanori; Goto, Akihiko; Amano, Nobuyuki


    DDI could be caused by the inhibition of OATP-mediated hepatic uptakes. The aim of this study is to set the risk criteria for the compounds that would cause DDI via OATP inhibition at the drug discovery stage. The IC50 values of OATP inhibitors for human OATP-mediated atorvastatin uptake were evaluated in the expression system. In order to set the risk criteria for OATP inhibition, the relationship was clarified between OATP inhibitory effect and severe adverse effects of OATP substrates, rhabdomyolysis, hyperbilirubinemia and jaundice. Rhabdomyolysis would be caused in the atorvastatin AUC more than 9-fold of that at a minimum therapeutic dose. The atorvastatin AUC was 6- to 9-fold increased with the OATP inhibitors of which IC50 values were ≤1 μmol/L. Hyperbilirubinemia and jaundice would be caused with the OATP inhibitors of which IC50 values were ≤6 μmol/L. This investigation showed that the compounds with IC50 of ≤1 μmol/L would have high risk for OATP-mediated DDI that would cause severe side effects. Before the detailed analysis based on the dosage, unbound fraction in blood and effective concentration to evaluate the clinical DDI potency, this criteria enable high throughput screening and optimize lead compounds at the drug discovery stage.

  13. Suppression of dynamin GTPase decreases α-synuclein uptake by neuronal and oligodendroglial cells: a potent therapeutic target for synucleinopathy

    Directory of Open Access Journals (Sweden)

    Konno Masatoshi


    Full Text Available Abstract Background The intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration. Results In this study, we have demonstrated that α-synuclein (αSYN, a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular αSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, αSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process. Conclusions Our findings shed light on the mode of αSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.

  14. BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aβ rise in human neuroblastoma SH-SY5Y cells

    Directory of Open Access Journals (Sweden)

    Edlund Michael


    Full Text Available Abstract Background Accumulation of amyloid β-peptide (Aβ in the plaques is one of the major pathological features in Alzheimer's disease (AD. Sequential cleavage of amyloid precursor protein (APP by β-site APP cleaving enzyme 1 (BACE-1 and γ-secretase results in the formation of Aβ peptides. Preventing Aβ formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development. Methods Combining BACE-1 and γ-secretase inhibition on Aβ secretion from human neuroblastoma SH-SY5Y cells was evaluated in this study. Secreted Aβ40 and Aβ42 levels were measured from SH-SY5Y cells stably transfected with APPwt or APPswe genes. A selective BACE inhibitor and the γ-secretase inhibitor LY450139 (semagacestat were used to inhibit respective secretase. Results LY450139 increased Aβ40 and Aβ42 secretion from SH-SY5Y APPwt cells at low concentrations (by 60% at 3 nM followed by subsequent inhibition at higher concentrations (IC50 90 nM. Washout studies showed that the Aβ increase evoked by 3 nM LY450139 was not due to enhanced cleavage following substrate accumulation but rather to activation of Aβ formation. By contrast, LY450139 inhibited Aβ formation from SH-SY5Y APPswe in a monophasic manner (IC50 18 nM. The BACE inhibitor per se inhibited Aβ secretion from both SH-SY5Y APPwt and SH-SY5Y APPswe cells with IC50s ranging between 7 - 18 nM and also prevented the increased Aβ secretion evoked by 3 nM LY450139. Combining the BACE inhibitor with higher inhibitory concentrations of LY450139 failed to demonstrate any clear additive or synergistic effects. Conclusion BACE-1 inhibition attenuates the Aβ increase evoked by LY450139 while not providing any obvious synergistic effects on LY450139-mediated inhibition.

  15. Research on Inhibition Effect of Multi-function Pickling Corrosion Inhibitor%多功能酸洗缓蚀剂缓蚀效果的研究

    Institute of Scientific and Technical Information of China (English)

    张晓冬; 喻果; 徐飞


    Hydrochloric acid pickling, sulfuric acid pickling, nitric acid pickling corrosion experiments of multifunctional pickling corrosion inhibitor developed by our company were carried out. The experimental results show that the multifunctional pickling corrosion inhibitor in hydrochloric acid pickling, sulfuric acid, nitric acid pickling process has very good inhibition effect for carbon steel and16 mn steel; different drug concentration has different corrosion inhibition effect. Multifunctional pickling corrosion inhibitor concentration commonly is 3‰~5‰, 10% hydrochloric acid pickling corrosion inhibition rate can reach 98.8%, 10% sulfuric acid pickling corrosion inhibition rate can reach 99.8%, 10% nitric acid pickling corrosion inhibition rate can reach 77%.%对威海翔宇环保科技股份有限公司研发的多功能酸洗缓蚀剂进行了盐酸酸洗、H2SO4 酸洗、硝酸酸洗缓蚀实验,实验结果表明,多功能酸洗缓蚀剂在盐酸酸洗、H2SO4酸洗、硝酸酸洗过程中对碳钢、16mn 钢都有很好的缓蚀效果,加药浓度不同,缓蚀效果不同.多功能酸洗缓蚀剂的加药浓度一般在 3‰~5‰,10%的盐酸酸洗的缓蚀率可达到 98.8%、10%的 H2SO4酸洗的缓蚀率可达到 99.8%、10%的硝酸酸洗的缓蚀率可达到 77%以上.

  16. Inhibiting properties and adsorption of an amine based fatty acid corrosion inhibitor on carbon steel in aqueous carbon dioxide solutions

    Energy Technology Data Exchange (ETDEWEB)

    Buchweishaija, Joseph


    Carbon dioxide corrosion is a major corrosion problem in oil and gas production systems and many organic inhibitors have been tested and used to protect the substrate from corrosion. This thesis studies the mechanism of interaction of the inhibitor molecule with the metallic substrate and how this affects the dissolution rate of the metal. The performance of a commercial amine based fatty acid corrosion inhibitor has been investigated using rotating cylinder electrodes and carbon steel electrodes in CO{sub 2} saturated formation water in the temperature range between 35 to 80{sup o}C. The corrosion process was monitored by electrochemical impedance measurements, and at the end of each experiment full polarization curves were recorded. When the inhibitor was applied on noncorroded electrodes, high inhibitor performance, over 99.7%, was observed independent of temperature. On precorroded electrodes inhibitor performance was found to depend on temperature and time of precorrosion. Above 60{sup o}C, the inhibitor performance decreased with increasing time of precorrosion, presumably because of the formation of a corrosion film of either iron carbonate or a combination of iron carbonate and iron carbide which prevent the inhibitor from reaching the surface. The inhibitor protection efficiency was assumed to be associated with the degree of inhibitor coverage at the material surface, and adsorption isotherms have been calculated in the concentration range between 0.1 ppm and 100 ppm. A Langmuir isotherm was found to give the best fit. The inhibitor performance on a 2 days precorroded rotating electrode was investigated at different solution pH ranging between 4.5 and 6.5 at 35{sup o}C. 130 refs., 80 figs., 22 tabs.

  17. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Backues, Steven K. [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States); Bednarek, Sebastian Y., E-mail: [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States)


    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  18. LeoA, B and C from Enterotoxigenic Escherichia coli (ETEC) Are Bacterial Dynamins

    DEFF Research Database (Denmark)

    Michie, Katharine A; Boysen, Anders; Low, Harry H


    C suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through...... membrane vesicle associated toxin secretion.......Escherichia coli (ETEC) strain H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify Leo...

  19. The Crystal Structure of BRAF in Complex with an Organoruthenium Inhibitor Reveals a Mechanism for Inhibition of an Active Form of BRAF Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Peng; Streu, Craig; Qin, Jie; Bregman, Howard; Pagano, Nicholas; Meggers, Eric; Marmorstein, Ronen (Wistar); (UPENN)


    Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF{sup V600E}, have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, based on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF{sup V600E} oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF{sup V600E} oncogene selective BRAF inhibitors for therapeutic application.

  20. Calpain Inhibitor Reduces Cancer-induced Bone Pain Possibly Through Inhibition of Osteoclastogenesis in Rat Cancer-induced Bone Pain Model

    Institute of Scientific and Technical Information of China (English)

    Jia-Ying Xu; Yu Jiang; Wei Liu; Yu-Guang Huang


    Background:Calpain,a calcium-dependent cysteine protease,has been demonstrated to regulate osteoclastogenesis,which is considered one of the major reasons for cancer-induced bone pain (CIBP).In the present study,calpain inhibitor was applied in a rat CIBP model to determine whether it could reduce CIBP through regulation of osteoclastogenesis activity.Methods:A rat CIBP model was established with intratibial injection of Walker 256 cells.Then,the efficacy of intraperitoneal administered calpain inhibitor Ⅲ (MDL28170,1 mg/kg) on mechanical withdrawal threshold (MWT) of bilateral hind paws was examined on postoperative days (PODs) 2,5,8,11,and 14.On POD 14,the calpain inhibitor's effect on tumor bone tartrate-resistant acid phosphatase (TRAP) stain and radiology was also carefully investigated.Results:Pain behavioral tests in rats showed that the calpain inhibitor effectively attenuated MWTs of both the surgical side and contralateral side hind paws on POD 5,8,and 11 (P < 0.05).TRAP-positive cell count of the surgical side bone was significantly decreased in the calpain inhibitor group compared with the vehicle group (P < 0.05).However,bone resorption and destruction measured by radiographs showed no difference between the two groups.Conclusions:Calpain inhibitor can effectively reduce CIBP of both the surgical side and nonsurgical side after tumor injection in a rat CIBP model.It may be due to the inhibition of receptor activator of nuclear factor-kappa B ligand-induced osteoclastogenesis.Whether a calpain inhibitor could be a novel therapeutic target to treat CIBP needs further investigation.

  1. Functional characterisation and Mutational analysis of a bacterial dynamin-like protein, DynA



    Membrane remodeling is a dynamic process that occurs in bacterial cells to facilitate substrate transport and to provide protection to bacteria during environmental stress. In eukaryotic cells, membrane remodeling is carried out by dynamin-like proteins (DLPs). These proteins are involved in diverse membrane-associated functions such as cargo transport via vesicles, cytokinesis, division of cell organelles and resistance to pathogens. DLPs are also conserved in bacterial species; howeve...

  2. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing


    Trochet, Delphine; Prudhon, Bernard; Jollet, Arnaud; Lorain, Stéphanie; Bitoun, Marc


    International audience; Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centron...

  3. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing


    Trochet, Delphine; Prudhon, Bernard; Jollet, Arnaud; Lorain, Stéphanie; Bitoun, Marc


    Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In orde...

  4. Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy


    Fraysse, Bodvaël; Guicheney, Pascale; Bitoun, Marc


    ABSTRACT Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2 R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atro...

  5. Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy


    Bodvaël Fraysse; Pascale Guicheney; Marc Bitoun


    International audience; Autosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2 R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively deve...

  6. Dynamin Forms a Src Kinase–sensitive Complex with Cbl and Regulates Podosomes and Osteoclast Activity



    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytos...

  7. Imaging single endocytic events reveals diversity in clathrin, dynamin and vesicle dynamics. (United States)

    Mattheyses, Alexa L; Atkinson, Claire E; Simon, Sanford M


    The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.

  8. Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

    Directory of Open Access Journals (Sweden)

    Julia Wenger

    Full Text Available Human dynamin-1-like protein (DNM1L is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.

  9. Regulation of cargo-selective endocytosis by dynamin 2 GTPase-activating protein girdin. (United States)

    Weng, Liang; Enomoto, Atsushi; Miyoshi, Hiroshi; Takahashi, Kiyofumi; Asai, Naoya; Morone, Nobuhiro; Jiang, Ping; An, Jian; Kato, Takuya; Kuroda, Keisuke; Watanabe, Takashi; Asai, Masato; Ishida-Takagishi, Maki; Murakumo, Yoshiki; Nakashima, Hideki; Kaibuchi, Kozo; Takahashi, Masahide


    In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

  10. A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD inhibits phosphorylation of CdsD and suppresses bacterial replication

    Directory of Open Access Journals (Sweden)

    Bulir David C


    Full Text Available Abstract Background We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. Results Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB, intermediate bodies (IB and reticulate bodies (RB, but lacked persistent bodies (PB, indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. Conclusion We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.

  11. Administration of PDE4 Inhibitors Suppressed the Pannus-Like Inflammation by Inhibition of Cytokine Production by Macrophages and Synovial Fibroblast Proliferation

    Directory of Open Access Journals (Sweden)

    Ichiro Miki


    Full Text Available A marked proliferation of synovial fibroblasts in joints leads to pannus formation in rheumatoid arthritis (RA. Various kinds of cytokines are produced in the pannus. The purpose of this study is to elucidate the effects of phosphodiesterase 4 (PDE4 inhibitors in a new animal model for the evaluation of pannus formation and cytokine production in the pannus. Mice sensitized with methylated bovine serum albumin (mBSA were challenged by subcutaneous implantation of a membrane filter soaked in mBSA solution in the back of the mice. Drugs were orally administered for 10 days. The granuloma formed around the filter was collected on day 11. It was chopped into pieces and cultured in vitro for 24 hr. The cytokines were measured in the supernatants. The type of cytokines produced in the granuloma was quite similar to those produced in pannus in RA. Both PDE4 inhibitors, KF66490 and SB207499, suppressed the production of IL-1β, TNF-α, and IL-12, and the increase in myeloperoxidase activity, a marker enzyme for neutrophils and hydroxyproline content. Compared to leflunomide, PDE4 inhibitors more strongly suppressed IL-12 production and the increase in myeloperoxidase activity. PDE4 inhibitors also inhibited lipopolysaccharide-induced TNF-α and IL-12 production from thioglycolate-induced murine peritoneal macrophages and the proliferation of rat synovial fibroblasts. These results indicate this model makes it easy to evaluate the effect of drugs on various cytokine productions in a granuloma without any purification step and may be a relevant model for evaluating novel antirheumatic drugs on pannus formation in RA. PDE4 inhibitors could have therapeutic effects on pannus formation in RA by inhibition of cytokine production by macrophages and synovial fibroblast proliferation.

  12. Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

    Directory of Open Access Journals (Sweden)

    Jayanthan Aarthi


    Full Text Available Abstract Background Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. Results AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. Conclusions In addition to previously described involvement of insulin like growth factor (IGF family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

  13. Biological activities of C1 inhibitor independent of protease inhibition%C1酯酶抑制剂的非蛋白酶抑制功能

    Institute of Scientific and Technical Information of China (English)

    杨晓凤; 张俊平; 胡振林


    C1 esterase inhibitor belongs to the superfamily of serine proteinase inhibitors (serpins) and regulates several important systems including the complement system, the contact activation system, the fibrinolytic system, and the intrinsic pathway of coagulation. It is currently used for the treatment of hereditary angioedema in clinic. However, recent studies have suggested that Cl esterase inhibitor may have potential use in the treatment of diseases other than hereditary angioedema, such as sepsis and reperfusion of ischemic myocardium, due to its new biological activities, for instance, anti-inflammation and anti-apoptosis, unrelated to protease inhibition. Here we review the biological activities of Cl inhibitor independent of protease inhibition.%C1酯酶抑制剂(C1 esterase inhibitor,C1INH)属于丝氨酸蛋白酶抑制剂家族,能够调节补体系统、激肽释放系统、纤溶系统和凝血系统.目前在临床号主要用于遗传性血管性水肿的治疗.但最近的研究表明C1 INH除丝氨酸蛋白酶抑制作用外,还具有多种非蛋白酶抑制功能,如抗炎和抗凋亡作用.而且很多动物实验和临床试验显示C1INH对脓毒症(sepsis),心肌缺血等疾病也有治疗作用.本文主要综述C1INH的非蛋白酶抑制功能的最新研究进展.

  14. In vitro anti-osteoclastogenic activity of p38 inhibitor doramapimod via inhibiting migration of pre-osteoclasts and NFATc1 activity

    Directory of Open Access Journals (Sweden)

    Seong-Hee Moon


    Full Text Available The mitogen activated protein kinase p38 plays a role in the receptor activator of NF-ĸB ligand (RANKL-induced osteoclast differentiation. In this study, we investigated the effect of p38 inhibitor doramapimod on the osteoclast differentiation. Doramapimod significantly inhibited the osteoclastogenesis of bone marrow macrophages (BMMs via attenuating the activation of p38 induced by M-CSF and RANKL. Importantly, doramapimod blocked the migration and fusion in pre-osteoclasts via the down-regulating NFATc1. The inhibitory effect of doramapimod on the migration/fusion of pre-osteoclasts via inhibiting NFATc1 activity were confirmed by measuring NFATc1 luciferase activity and evaluating the mRNA expression of NFATc1-responsive genes related to the osteoclastic migration/fusion. These results suggested anti-osteoclastogenic activity of doramapimod via inhibiting migration/fusion of pre-osteoclasts and NFATc1 activity.

  15. Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma. (United States)

    Fishburn, C S; Slaney, J M; Carman, R J; Curtis, M A


    The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.

  16. Anti-HIV cytotoxicity enzyme inhibition and molecular docking studies of quinoline based chalcones as potential non-nucleoside reverse transcriptase inhibitors (NNRT). (United States)

    Hameed, Asima; Abdullah, Muhammad Imran; Ahmed, Ejaz; Sharif, Ahsan; Irfan, Ahmad; Masood, Sara


    A series of fourteen (A1 - A14) qunioline based chalcones were screened for reverse transcriptase inhibitors (RT) and found potentially active against RT. Bioassay, theoretical and dockings studies with RT (the enzyme required for reverse transcription of viral RNA) results showed that the type and positions of the substituents seemed to be critical for their inhibition against RT. The bromo and chloro substituted chalcone displayed high degree of inhibition against RT. The A4 andA6 showed high interaction with RT, contributing high free binding energy (ΔG -9.30 and -9.13kcal) and RT inhibition value (IC50 0.10μg/ml and 0.11μg/ml).

  17. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)


    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  18. NNC 55-0396, a T-type Ca2+ channel inhibitor, inhibits angiogenesis via suppression of hypoxia-inducible factor-1α signal transduction. (United States)

    Kim, Ki Hyun; Kim, Dongyoung; Park, Ju Yeol; Jung, Hye Jin; Cho, Yong-Hee; Kim, Hyoung Kyu; Han, Jin; Choi, Kang-Yell; Kwon, Ho Jeong


    Mitochondrial respiration is required for hypoxia-inducible factor (HIF)-1α stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Herein, small molecules that inhibit HIF-1α protein stability by targeting mitochondrial energy production were screened using the Library of Pharmacologically Active Compounds and cell growth assay in galactose or glucose medium. NNC 55-0396, a T-type Ca(2+) channel inhibitor, was selected as a hit from among 1,280 small molecules. NNC 55-0396 suppressed mitochondrial reactive oxygen species-mediated HIF-1α expression as well as stabilization by inhibiting protein synthesis in a dose-dependent manner. NNC 55-0396 inhibited tumor-induced angiogenesis in vitro and in vivo by suppressing HIF-1α stability. Moreover, NNC 55-0396 significantly suppressed glioblastoma tumor growth in a xenograft model. Thus, NNC 55-0396, a small molecule targeting T-type Ca(2+) channel, was identified by the systemic cell-based assay and was shown to have antiangiogenic activity via the suppression of HIF-1α signal transduction. These results provide new insights into the biological network between ion channel and HIF-1α signal transduction. HIF-1α overexpression has been demonstrated in hypoxic cancer cells. NNC 55-0396, a T-type Ca(2+) channel inhibitor, inhibited HIF-1α expression via both proteasomal degradation and protein synthesis pathways. T-type Ca(2+) channel inhibitors block angiogenesis by suppressing HIF-1α stability and synthesis. NNC 55-0396 could be a potential therapeutic drug candidate for cancer treatment.

  19. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar


    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  20. Similarly potent inhibition of adenylyl cyclase by P-site inhibitors in hearts from wild type and AC5 knockout mice.

    Directory of Open Access Journals (Sweden)

    Joerg H Braeunig

    Full Text Available Adenylyl cyclase type 5 (AC5 was described as major cardiac AC isoform. The knockout of AC5 (AC5KO exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine and NKY80 [2-amino-7-(2-furanyl-7,8-dihydro-5(6H-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold. These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.

  1. Inhibition effects of PMA/SbBr3 complex inhibitor on copper and copper-nickel alloy in LiBr solutions

    Institute of Scientific and Technical Information of China (English)

    HU Xian-qi; LIANG Cheng-hao; HUANG Nai-bao


    The effects of PMA/SbBr3 inhibitor on copper and copper-nickel alloy in 55%LiBr solution were investigated by chemical immersion and electrochemical measurements. The results indicate that in boiling 55% LiBr solution containing PMA/SbBr3 inhibitor, corrosion rates of copper and copper-nickel alloy are 67.48 μm/a and 38. 14μm/a, respectively. Since both anodic and cathodic electrochemical reactions can be inhibited, PMA/SbBr3 belongs to complex inhibitor. PMA has the effect of inhibiting hydrogen evolution and [PMo12 O40]3- , the anion of PMA,has a strong oxidizing effect. Sb3+ also shows an oxidizing effect. It may exist in LiBr solutions stably with PMA.Because of the synergistic effect of PMA and Sb3+ , a protective film, comprising CuO, Cu2O and Sb, formed on copper and copper-nickel alloy surface may prevent Br- from diffusing to the surface of metals. As a result, the anticorrosion performance of copper and copper-nickel alloy may be improved.

  2. Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization. (United States)

    Boppana, Nithin B; Kodiha, Mohamed; Stochaj, Ursula; Lin, Ho-sheng; Haimovitz-Friedman, Adriana; Bielawska, Alicja; Bielawski, Jacek; Divine, George W; Boyd, John A; Korbelik, Mladen; Separovic, Duska


    The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

  3. Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis. (United States)

    Cianchetta, Giovanni; Stouch, Terry; Yu, Wangsheng; Shi, Zhi-Cai; Tari, Leslie W; Swanson, Ronald V; Hunter, Michael J; Hoffman, Isaac D; Liu, Qingyun


    Trytophan Hydroxylase Type I (TPH1), most abundantly expressed in the gastrointestinal tract, initiates the synthesis of serotonin by catalyzing hydroxylation of tryptophan in the presence of biopterin and oxygen. We have previously described three series of novel, periphery-specific TPH1 inhibitors that selectively deplete serotonin in the gastrointestinal tract. We have now determined co-crystal structures of TPH1 with three of these inhibitors at high resolution. Analysis of the structural data showed that each of the three inhibitors fills the tryptophan binding pocket of TPH1 without reaching into the binding site of the cofactor pterin, and induces major conformational changes of the enzyme. The enzyme-inhibitor complexes assume a compact conformation that is similar to the one in tryptophan complex. Kinetic analysis showed that all three inhibitors are competitive versus the substrate tryptophan, consistent with the structural data that the compounds occupy the tryptophan binding site. On the other hand, all three inhibitors appear to be uncompetitive versus the cofactor 6-methyltetrahydropterin, which is not only consistent with the structural data but also indicate that the hydroxylation reaction follows an ordered binding mechanism in which a productive complex is formed only if tryptophan binds only after pterin, similar to the kinetic mechanisms of tyrosine and phenylalanine hydroxylase.

  4. Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580. (United States)

    Conway, James G; McDonald, Brad; Parham, Janet; Keith, Barry; Rusnak, David W; Shaw, Eva; Jansen, Marilyn; Lin, Peiyuan; Payne, Alan; Crosby, Renae M; Johnson, Jennifer H; Frick, Lloyd; Lin, Min-Hwa Jasmine; Depee, Scott; Tadepalli, Sarva; Votta, Bart; James, Ian; Fuller, Karen; Chambers, Timothy J; Kull, Frederick C; Chamberlain, Stanley D; Hutchins, Jeff T


    Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

  5. Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein

    Directory of Open Access Journals (Sweden)

    Xing Lin


    Full Text Available Background: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. Methods: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. Results: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. Conclusion: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.

  6. The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis (United States)

    Wu, Wenman; Li, Hongbo; Navaneetham, Duraiswamy; Reichenbach, Zachary W.; Tuma, Ronald F.


    Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl3-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy. PMID:22674803

  7. FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1). (United States)

    Dombrowski, Christian; Helledie, Torben; Ling, Ling; Grünert, Martin; Canning, Claire A; Jones, C Michael; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M


    Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.

  8. Combination Treatment with PPARγ Ligand and Its Specific Inhibitor GW9662 Downregulates BIS and 14-3-3 Gamma, Inhibiting Stem-Like Properties in Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Chang-Nim Im


    Full Text Available PPARγ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPARγ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA transfection with PPARγ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose polymerase (PARP cleavage. Taken together, our findings suggest that a combination therapy using PPARγ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.

  9. Itraconazole, a P-glycoprotein and CYP3A4 inhibitor, markedly raises the plasma concentrations and enhances the renin-inhibiting effect of aliskiren. (United States)

    Tapaninen, Tuija; Backman, Janne T; Kurkinen, Kaisa J; Neuvonen, Pertti J; Niemi, Mikko


    In a randomized crossover study, 11 healthy volunteers took 100 mg (first dose 200 mg) of the antifungal drug itraconazole, a P-glycoprotein and CYP3A4 inhibitor, or placebo twice daily for 5 days. On day 3, they ingested a single 150-mg dose of aliskiren, a renin inhibitor used in the treatment of hypertension. Itraconazole raised the peak plasma aliskiren concentration 5.8-fold (range, 1.1- to 24.3-fold; P plasma aliskiren concentration-time curve 6.5-fold (range, 2.6- to 20.5-fold; P Plasma renin activity 24 hours after aliskiren intake was 68% lower during the itraconazole phase than during the placebo phase (P = .011). In conclusion, itraconazole markedly raises the plasma concentrations and enhances the renin-inhibiting effect of aliskiren. The interaction is probably mainly explained by inhibition of the P-glycoprotein-mediated efflux of aliskiren in the small intestine, with a minor contribution from inhibition of CYP3A4. Concomitant use of aliskiren and itraconazole is best avoided.

  10. N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells. (United States)

    Medina-Enríquez, Miriam Marlene; Alcántara-Farfán, Verónica; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José Guadalupe; Rodríguez-Páez, Lorena; Vargas-Ramírez, Alba Laura


    Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.

  11. Inhibition of Drp1-dependent mitochondrial fragmentation and apoptosis by a polypeptide antagonist of calcineurin (United States)

    Cereghetti, GM; Costa, V; Scorrano, L


    During apoptosis, mitochondria lose their membrane potential and undergo fragmentation around the time of release of cytochrome c. Apoptotic fission is at least in part sustained by the translocation of dynamin-related protein 1 (Drp1), normally located in the cytosol, to mitochondria. This process depends on dephosphorylation of Drp1 by the phosphatase calcineurin. Here, we report the identification of a novel inhibitor of this process. A polypeptide (PPD1) from the immunophilin FKBP52 inhibits calcineurin activation triggered by mitochondrial dysfunction. PPD1 blocks Drp1 translocation to mitochondria and fragmentation of the organelle. PPD1 delays apoptosis by intrinsic stimuli by preventing fragmentation and release of cytochrome c. Cells expressing PPD1 display enhanced clonogenic ability after exposure to staurosporine. A genetic analysis revealed that the activity of PPD1 is independent of the BH3-only protein BAD, another target of calcineurin during apoptosis, and is not additive to inhibition of Drp1. Thus, PPD1 is a novel inhibitor of apoptosis that elucidates the function of calcineurin-dependent mitochondrial fragmentation in the amplification of cell death. PMID:20489733

  12. Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels. (United States)

    Yarotskyy, V; Elmslie, K S


    L-type calcium channels (Ca((V))1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca((V))1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca((V))1.2 channels. Ca((V))1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca((V))1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC(50)=29.5+/-12 microM, which is close to that for slow activation and inhibition. We conclude that roscovitine binds to an extracellular site on Ca((V))1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.

  13. Lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, downregulates thymidylate synthase by inhibiting the nuclear translocation of EGFR and HER2.

    Directory of Open Access Journals (Sweden)

    Hwang-Phill Kim

    Full Text Available BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS, which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib. CONCLUSIONS AND SIGNIFICANCE: These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.

  14. Characterization of Long-Lasting Oatp Inhibition by Typical Inhibitor Cyclosporine A and In Vitro-In Vivo Discrepancy in Its Drug Interaction Potential in Rats. (United States)

    Taguchi, Takayuki; Masuo, Yusuke; Kogi, Tatsuya; Nakamichi, Noritaka; Kato, Yukio


    Quantitative assessment of potential drug-drug interactions (DDIs) is one of the major focuses in drug development. The aim of the present study was to quantitatively evaluate in vitro-in vivo discrepancy of DDI potential for prototypical organic anion transporting polypeptide (Oatp) inhibitor cyclosporine A (CsA) using rats. Plasma concentration of pravastatin, prototypical Oatp substrate, after oral administration was increased by CsA intravenously administered at 1 d before the pravastatin administration. The ratio of the area under the curve of pravastatin to the control was much higher than the R-values calculated using the plasma unbound concentrations of CsA and the inhibition constant (Ki) assessed in isolated hepatocytes, indicating in vitro-in vivo discrepancy. This interaction with pravastatin persisted for 3 d after CsA administration, demonstrating long-lasting inhibition in vivo. The Ki value for unbound CsA in the presence of serum was comparable with that in its absence. M1, the major metabolite of CsA inhibited pravastatin uptake at much higher concentration compared with its plasma unbound concentration. Thus, the DDI potential of CsA-mediated hepatic Oatp inhibition cannot be extrapolated from in vitro data, and this could be due to the long-lasting Oatp inhibition by CsA, but not the effect of plasma protein or metabolites.

  15. Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

    Energy Technology Data Exchange (ETDEWEB)

    Shenoy, Rajesh T. [National Univ. of Singapore (Singapore); Thangamani, Saravanan [National Univ. of Singapore (Singapore); Univ. of Texas Medical Branch, Galveston, TX (United States); Velazquez-Campoy, Adrian [Univ. of Zaragoza (Spain); Ho, Bow [National Univ. of Singapore (Singapore); Ding, Jeak Ling [National Univ. of Singapore (Singapore); Sivaraman, J. [National Univ. of Singapore (Singapore); Kursula, Petri [Univ. of Oulu (Germany)


    Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki=1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

  16. Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate. (United States)

    Pestel, Sabine; Nath, Annegret; Jungermann, Kurt; Schieferdecker, Henrike L


    Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.

  17. Carbonic anhydrase inhibitors. Inhibition of the human cytosolic isoforms I and II and transmembrane, tumor-associated isoforms IX and XII with boronic acids. (United States)

    Winum, Jean-Yves; Innocenti, Alessio; Scozzafava, Andrea; Montero, Jean-Louis; Supuran, Claudiu T


    A series of aromatic, arylalkenyl- and arylalkyl boronic acids were assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC isoforms, the cytosolic human (h) hCA I and II, and the transmembrane, tumor-associated hCA IX and XII. The best hCA I and II inhibitor was biphenyl boronic acid with, a K(I) of 3.7-4.5 microM, whereas the remaining derivatives showed inhibition constants in the range of 6.0-1560 microM for hCA I and of 6.0-1050 microM for hCA II, respectively. hCA IX and XII were effectively inhibited by most of the aromatic boronic acids (K(I)s of 7.6-12.3 microM) whereas the arylalkenyl and aryl-alkyl derivatives generally showed weaker inhibitory properties (K(I)s of 34-531 microM). The nature of the moiety substituting the boronic acid group strongly influenced the CA inhibitory activity, with inhibitors possessing low micromolar to millimolar activity being detected in this small series of investigated compounds. This study proves that the B(OH)(2) moiety represents a new zinc-binding group for the generation of effective CA inhibitors targeting isoforms with medicinal chemistry applications. The boronic acids probably bind to the Zn(II) ion within the CA active site leading to a tetrahedral geometry of the metal ion and of the B(III) derivative.

  18. Synergistic regulation of glutamatergic transmission by serotonin and norepinephrine reuptake inhibitors in prefrontal cortical neurons. (United States)

    Yuen, Eunice Y; Qin, Luye; Wei, Jing; Liu, Wenhua; Liu, Aiyi; Yan, Zhen


    The monoamine system in the prefrontal cortex has been implicated in various mental disorders and has been the major target of anxiolytics and antidepressants. Clinical studies show that serotonin and norepinephrine reuptake inhibitors (SNRIs) produce better therapeutic effects than single selective reuptake inhibitors, but the underlying mechanisms are largely unknown. Here, we found that low dose SNRIs, by acting on 5-HT(1A) and α2-adrenergic receptors, synergistically reduced AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents and AMPAR surface expression in prefrontal cortex pyramidal neurons via a mechanism involving Rab5/dynamin-mediated endocytosis of AMPARs. The synergistic effect of SNRIs on AMPARs was blocked by inhibition of activator of G protein signaling 3, a G protein modulator that prevents reassociation of G(i) protein α subunit and prolongs the βγ-mediated signaling pathway. Moreover, the depression of AMPAR-mediated excitatory postsynaptic currents by SNRIs required p38 kinase activity, which was increased by 5-HT(1A) and α2-adrenergic receptor co-activation in an activator of G protein signaling 3-dependent manner. These results have revealed a potential mechanism for the synergy between the serotonin and norepinephrine systems in the regulation of glutamatergic transmission in cortical neurons.

  19. Inhibition of NOS-2 induction in LPS-stimulated J774.2 cells by 1, 5-isoquinolinediol, an inhibitor of PARP. (United States)

    Olszanecki, R; Gebska, A; Jawień, J; Jakubowski, A; Korbut, R


    Activation of both poly (ADP-ribose) polymerase (PARP) and inducible nitric oxide synthase (NOS-2) have been implicated in the pathogenesis of various forms of inflammation, therefore compounds which may simultaneously inhibit both pathways are of potential therapeutic interest. We tested the influence of potent inhibitor of PARP, 1, 5-isoquinolinediol (ISO), on NOS-2 induction in model of mouse macrophages (cell line J774.2) stimulated with lipopolysaccharide (1 microg/ml). Pretreatment with ISO (1-300 microM) resulted in dose-dependent inhibition of accumulation of NOS-2-derived nitrite in culture medium (IC(50) = 9,3 microM) as well as inhibition of NOS-2 protein induction in cultured J774.2 cells; ISO given 10 hours after LPS did not influence activity of NOS-2. Interestingly, another PARP inhibitor, 3-aminobenzamide (3-AB, 10-3000 microM), did not influence 24-hr nitrite accumulation in J774.2 cell culture, either administered 15 minutes prior to LPS or 10 hrs after LPS. Scavenging of reactive oxygen species by use of mixture of SOD and catalase (SOD/Cat, 100/300 - 1000/3000 U/ml) as well as cell permeable SOD-mimetic [Mn(III)TBAP, 1- 100 microM], did not influence NOS-2 induction in J774.2 cells. In summary, we identified 1, 5-isoquinoline as potent inhibitor of induction of NOS-2 in LPS-treated mouse macrophages. The exact mechanism of inhibitory action of this compound on NOS-2 induction requires further investigation.

  20. Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode. (United States)

    Sawatzky, Edgar; Wehle, Sarah; Kling, Beata; Wendrich, Jan; Bringmann, Gerhard; Sotriffer, Christoph A; Heilmann, Jörg; Decker, Michael


    Butyrylcholinesterase (BChE) is a promising target for the treatment of later stage cognitive decline in Alzheimer's disease. A set of pseudo-irreversible BChE inhibitors with high selectivity over hAChE was synthesized based on carbamates attached to tetrahydroquinazoline scaffolds with the 2-thiophenyl compound 2p as the most potent inhibitor of eqBChE (KC = 14.3 nM) and also of hBChE (KC = 19.7 nM). The inhibitors transfer the carbamate moiety onto the active site under release of the phenolic tetrahydroquinazoline scaffolds that themselves act as neuroprotectants. By combination of kinetic data with molecular docking studies, a plausible binding model was probed describing how the tetrahydroquinazoline scaffold guides the carbamate into a close position to the active site. The model explains the influence of the carrier scaffold onto the affinity of an inhibitor just before carbamate transfer. This strategy can be used to utilize the binding mode of other carbamate-based inhibitors.

  1. Inhibition mechanism analysis & research of the poly aspartic acid corrosion inhibitor%聚天冬氨酸缓蚀剂缓蚀机理分析研究

    Institute of Scientific and Technical Information of China (English)



    文中介绍了聚天冬氨酸缓蚀剂缓蚀机理,并对聚天冬氨酸缓蚀剂机理的研究现状以及发展趋势进行了综述。%It was introduced inhibition mechanism of the poly aspartic acid corrosion inhibitor. Mean- while, it was summarizeed the research progress of the inhibition meehanismf the poly aspartic acid cor- rosion inhibitor and the development trend of the poly aspartic acid corrosion inhibitor in this article.

  2. Acquired resistance of pancreatic cancer cells to treatment with gemcitabine and HER-inhibitors is accompanied by increased sensitivity to STAT3 inhibition. (United States)

    Ioannou, Nikolaos; Seddon, Alan M; Dalgleish, Angus; Mackintosh, David; Solca, Flavio; Modjtahedi, Helmout


    Drug-resistance is a major contributing factor for the poor prognosis in patients with pancreatic cancer. We have shown previously that the irreversible ErbB family blocker afatinib, is more effective than the reversible EGFR tyrosine kinase inhibitor erlotinib in inhibiting the growth of human pancreatic cancer cells. The aim of this study was to develop human pancreatic cancer cell (BxPc3) variants with acquired resistance to treatment with gemcitabine, afatinib, or erlotinib, and to investigate the molecular changes that accompany the acquisition of a drug-resistant phenotype. We also investigated the therapeutic potential of various agents in the treatment of such drug-resistant variants. Three variant forms of BxPc3 cells with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) were developed following treatment with increasing doses of such drugs. The expression level, mutational and phosphorylation status of various growth factor receptors and downstream cell signaling molecules were determined by FACS, human phopsho-RTK array, and western blot analysis while the sulforhodamine B assay was used for determining the effect of various agents on the growth of such tumours. We found that all three BxPc3 variants with acquired resistance to gemcitabine (BxPc3GEM), afatinib (BxPc3AFR) or erlotinib (BxPc3OSIR) also become less sensitive to treatment with the two other agents. Acquisition of resistance to these agents was accompanied by upregulation of p-c-MET, p-STAT3, CD44, increased autocrine production of EGFR ligand amphiregulin and differential activation status of EGFR tyrosine residues as well as downregulation of total and p-SRC. Of all therapeutic interventions examined, including the addition of an anti-EGFR antibody ICR62, an anti-CD44 monoclonal antibody, and of STAT3 or c-MET inhibitors, only treatment with the STAT3 inhibitor Stattic produced a higher growth inhibitory effect in all three drug-resistant variants

  3. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. (United States)

    Clarke, J F; Young, P W; Yonezawa, K; Kasuga, M; Holman, G D


    Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities. PMID:8010944

  4. Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling. (United States)

    Sattar, Rabia; Ali, Syed Abid; Kamal, Mustafa; Khan, Aftab Ahmed; Abbasi, Atiya


    Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.

  5. The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2* (United States)

    Su, Ya-Chi; Miller, Tara N.; Navaneetham, Duraiswamy; Schoonmaker, Robert T.; Sinha, Dipali; Walsh, Peter N.


    To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu98, Tyr143, Ile151, Arg3704, Lys192, and Tyr5901) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal Km values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of kcat for S-2366 hydrolysis. All six Ala mutants displayed deficient kcat values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of Ki except for K192A, and Y5901A, which displayed increased values of Ki. The integrity of the S1 binding site residue, Asp189, utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr143, Ile151, Arg3704, and Tyr5901) are important for S-2366 hydrolysis; Glu98 and Lys192 are essential for FIX but not S-2366 hydrolysis; and Lys192 and Tyr5901 are required for both inhibitor and macromolecular substrate interactions. PMID:21778227

  6. Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model. (United States)

    Gao, Yong-Jing; Cheng, Jen-Kun; Zeng, Qing; Xu, Zhen-Zhong; Decosterd, Isabelle; Xu, Xiaoyin; Ji, Ru-Rong


    Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

  7. In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors. (United States)

    Benvenuto, Monica; Masuelli, Laura; De Smaele, Enrico; Fantini, Massimo; Mattera, Rosanna; Cucchi, Danilo; Bonanno, Elena; Di Stefano, Enrica; Frajese, Giovanni Vanni; Orlandi, Augusto; Screpanti, Isabella; Gulino, Alberto; Modesti, Andrea; Bei, Roberto


    Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21-Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT-61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer.

  8. A Non-ATP-Competitive Dual Inhibitor of JAK2 and BCR-ABL Kinases: Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition. (United States)

    Jatiani, Shashidhar S; Cosenza, Stephen C; Reddy, M V Ramana; Ha, Ji Hee; Baker, Stacey J; Samanta, Ajoy K; Olnes, Matthew J; Pfannes, Loretta; Sloand, Elaine M; Arlinghaus, Ralph B; Reddy, E Premkumar


    Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

  9. Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors. (United States)

    Ducker, G S; Atreya, C E; Simko, J P; Hom, Y K; Matli, M R; Benes, C H; Hann, B; Nakakura, E K; Bergsland, E K; Donner, D B; Settleman, J; Shokat, K M; Warren, R S


    The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

  10. Blocking GSK3β-mediated dynamin1 phosphorylation enhances BDNF-dependent TrkB endocytosis and the protective effects of BDNF in neuronal and mouse models of Alzheimer's disease. (United States)

    Liu, Xiang-Hua; Geng, Zhao; Yan, Jing; Li, Ting; Chen, Qun; Zhang, Qun-Ye; Chen, Zhe-Yu


    Endocytosis of tropomyosin related kinase B (TrkB) receptors has critical roles in brain-derived neurotrophic factor (BDNF) mediated signal transduction and biological function, however the mechanism that is governing TrkB endocytosis is still not completely understood. In this study, we showed that GSK3β, a key kinase in neuronal development and survival, could regulate TrkB endocytosis through phosphorylating dynamin1 (Dyn1) but not dynamin2 (Dyn2). Moreover, we found that beta-amyloid (Aβ) oligomer exposure could impair BDNF-dependent TrkB endocytosis and Akt activation through enhancing GSK3β activity in cultured hippocampal neurons, which suggested that BDNF-induced TrkB endocytosis and the subsequent signaling were impaired in neuronal model of Alzheimer's disease (AD). Notably, we found that inhibiting GSK3β phosphorylating Dyn1 by using TAT-Dyn1SpS could rescue the impaired TrkB endocytosis and Akt activation upon BDNF stimuli under Aβ exposure. Finally, TAT-Dyn1SpS could facilitate BDNF-mediated neuronal survival and cognitive enhancement in mouse models of AD. These results clarified a role of GSK3β in BDNF-dependent TrkB endocytosis and the subsequent signaling, and provided a potential new strategy by inhibiting GSK3β-induced Dyn1 phosphorylation for AD treatment.

  11. NS-398, a selective cyclooxygenase-2 inhibitor, reduces experimental bladder carcinoma outgrowth by inhibiting tumor cell proliferation.

    NARCIS (Netherlands)

    Smakman, N.; Schaap, N.P.M.; Snijckers, C.M.; Rinkes, M.J.; Kranenburg, O.


    OBJECTIVES: To evaluate the efficacy of the selective cyclooxygenase-2 (COX-2) inhibitor NS-398 in treating experimental T24 bladder carcinoma, and to assess its effect on tumor cell proliferation and survival and tumor vascularization. COX-2 overexpression is frequently observed in bladder carcinom

  12. Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

    DEFF Research Database (Denmark)

    Thastrup, Ole; Knudsen, J B; Lemmich, J;


    Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity (Km = 1.1 microM) cAMP-phosphodiesterase from human...... platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant...

  13. Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Rebola, Nelson


    interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal...... neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given...

  14. Endophilin isoforms have distinct characteristics in interactions with N-type Ca2+ channels and dynamin

    Institute of Scientific and Technical Information of China (English)

    Qi Tian; Ji-Feng Zhang; Jinjin Fan; Zhihong Song; Yuan Chen


    Objective Formation of the endophilin Ⅱ-Ca2+ channel complex is Ca2+-dependent in clathrin-mediated endocytosis.However,little is known about whether the other two endophilin isoforms have the same features.The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca2+ channels and dynamin Ⅰ.Methods N-type Ca2+ channel C-terminal fragments (NCFs) synthesized with a 3H-leucine-labeled kit,were incubated with endophilin-GST fusion proteins,followed by pull-down assay.Results were counted on a scintillation counter.In addition,the different endophilin isoforms were each co-transfected with dynamin Ⅰ into 293T cells,followed by flow cytometry and co-immunoprecipitation assay.Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin Ⅰ.Results All three isoforms interacted with NCF.Endophilins Ⅰ and Ⅱ demonstrated clear Ca2+-dependent interactions with NCF,whereas endophilin Ⅲ did not.Co-immunoprecipitation showed that,compared to endophilin Ⅰ/Ⅱ,the interaction between endophilin Ⅲ and dynamin Ⅰ was significantly increased.Similar results were obtained from flow cytometry.Furthermore,endophilin Ⅲ had a higher overlap coefficient with dynamin Ⅰ in co-transfected 293T cells.Conclusion Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin Ⅰ.Endophilin Ⅲ binding to NCF is Ca2+-independent,implying that it plays a different role in clathrin-mediated endocytosis.

  15. The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, cell proliferation, multicellular development, and Cdk5 nuclear translocation in Dictyostelium discoideum. (United States)

    Huber, Robert J; O'Day, Danton H


    Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 µM and ≈ 50% rescued in 100 µM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However, roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development. Copyright © 2011 Wiley Periodicals, Inc.

  16. Investigation of Chalcones as Selective Inhibitors of the Breast Cancer Resistance Protein: Critical Role of Methoxylation in both Inhibition Potency and Cytotoxicity (United States)

    Valdameri, Glaucio; Gauthier, Charlotte; Terreux, Raphaël; Kachadourian, Rémy; Day, Brian J.; Winnischofer, Sheila M. B.; Rocha, Maria E. M.; Frachet, Véronique; Ronot, Xavier; Di Pietro, Attilio; Boumendjel, Ahcène


    ABCG2 plays a major role in anticancer-drug efflux and related tumor multidrug resistance. Potent and selective ABCG2 inhibitors with low cytotoxicity were investigated among a series of 44 chalcones and analogues (1,3-diarylpropenones), by evaluating their inhibitory effect on the transport of mitoxantrone, a known ABCG2 substrate. Six compounds producing complete inhibition with IC50 values below 0.5 µM and high selectivity for ABCG2 were identified. The number and position of methoxy substituents appeared to be critical for both inhibition and cytotoxicity. The best compounds, with potent inhibition and low toxicity, contained an N-methyl-1-indolyl (compound 38) or a 6′-hydroxyl-2′,4′-dimethoxy-1-phenyl (compound 27) moiety (A-ring) and two methoxy groups at positions 2 and 6 of the 3-phenyl moiety (B-ring). Methoxy substitution contributed to inhibition at positions 3 and 5, but had a negative effect at position 4. Finally, methoxy groups at positions 3, 4, and 5 of the B-ring markedly increased cytotoxicity and, therefore, should be avoided. PMID:22449016

  17. Inhibition of Leishmania (Leishmania amazonensis and rat arginases by green tea EGCG, (+-catechin and (--epicatechin: a comparative structural analysis of enzyme-inhibitor interactions.

    Directory of Open Access Journals (Sweden)

    Matheus Balduíno Goncalves dos Reis

    Full Text Available Epigallocatechin-3-gallate (EGCG, a dietary polyphenol (flavanol from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+-catechin and (--epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L and rat liver arginase (ARG-1. The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+-catechin and (--epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+-catechin (IC50 = 0.8 µM followed by (--epicatechin (IC50 = 1.8 µM, gallic acid (IC50 = 2.2 µM and EGCG (IC50 = 3.8 µM. Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC50 values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.

  18. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with novel Schiff bases: identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones. (United States)

    Sarikaya, Busra; Ceruso, Mariangela; Carta, Fabrizio; Supuran, Claudiu T


    A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.

  19. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    Energy Technology Data Exchange (ETDEWEB)

    Aoyagi-Scharber, Mika, E-mail: [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States); Gardberg, Anna S. [Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110 (United States); Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A. [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States)


    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

  20. Molecular dynamics simulations of the free and inhibitor-bound cruzain systems in aqueous solvent: insights on the inhibition mechanism in acidic pH. (United States)

    Hoelz, L V B; Leal, V F; Rodrigues, C R; Pascutti, P G; Albuquerque, M G; Muri, E M F; Dias, L R S


    The major cysteine protease of Trypanosoma cruzi, cruzain (CRZ), has been described as a therapeutic target for Chagas' disease, which affects millions of people worldwide. Thus, a series of CRZ inhibitors has been studied, including a new competitive inhibitor, Nequimed176 (NEQ176). Nevertheless, the structural and dynamic basis for CRZ inhibition remains unclear. Hoping to contribute to this ever-growing understanding of timescale dynamics in the CRZ inhibition mechanism, we have performed the first study using 100 ns of molecular dynamics (MD) simulations of two CRZ systems in an aqueous solvent under pH 5.5: CRZ in the apo form (ligand free) and CRZ complexed to NEQ176. According to the MD simulations, the enzyme adopts an open conformation in the apo form and a closed conformation in the NEQ176-CRZ complex. We also suggest that this closed conformation is related to the hydrogen-bonding interactions between NEQ176 and CRZ, which occurs through key residues, mainly Gly66, Met68, Asn69, and Leu160. In addition, the cross-correlation analysis shows evidence of the correlated motions among Ala110-Asp140, Leu160-Gly189, and Glu190-Gly215 subdomains, as well as, the movements related to Ala1-Thr59 and Asp60-Pro90 regions seem to be crucial for CRZ activity.

  1. PLGA-PEG Nanoparticles Coated with Anti-CD45RO and Loaded with HDAC Plus Protease Inhibitors Activate Latent HIV and Inhibit Viral Spread (United States)

    Tang, Xiaolong; Liang, Yong; Liu, Xinkuang; Zhou, Shuping; Liu, Liang; Zhang, Fujina; Xie, Chunmei; Cai, Shuyu; Wei, Jia; Zhu, Yongqiang; Hou, Wei


    Activating HIV-1 proviruses in latent reservoirs combined with inhibiting viral spread might be an effective anti-HIV therapeutic strategy. Active specific delivery of therapeutic drugs into cells harboring latent HIV, without the use of viral vectors, is a critical challenge to this objective. In this study, nanoparticles of poly(lactic-co-glycolic acid)-polyethylene glycol diblock copolymers conjugated with anti-CD45RO antibody and loaded with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and/or protease inhibitor nelfinavir (Nel) were tested for activity against latent virus in vitro. Nanoparticles loaded with SAHA, Nel, and SAHA + Nel were characterized in terms of size, surface morphology, zeta potential, entrapment efficiency, drug release, and toxicity to ACH-2 cells. We show that SAHA- and SAHA + Nel-loaded nanoparticles can target latently infected CD4+ T-cells and stimulate virus production. Moreover, nanoparticles loaded with SAHA + NEL were capable of both activating latent virus and inhibiting viral spread. Taken together, these data demonstrate the potential of this novel reagent for targeting and eliminating latent HIV reservoirs.

  2. miR-564 acts as a dual inhibitor of PI3K and MAPK signaling networks and inhibits proliferation and invasion in breast cancer (United States)

    Mutlu, Merve; Saatci, Özge; Ansari, Suhail A.; Yurdusev, Emre; Shehwana, Huma; Konu, Özlen; Raza, Umar; Şahin, Özgür