Systematic comparison of eight methods for preparation of high purity sulfated fucans extracted from the brown alga Pelvetia canaliculata.

Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans.

Design of new disubstituted imidazo[1,2-b]pyridazine derivatives as selective Haspin inhibitors. Synthesis, binding mode and anticancer biological evaluation.

Haspin is a mitotic protein kinase required for proper cell division by modulating Aurora B kinase localisation and activity as well as histone phosphorylation. Here a series of imidazopyridazines based on the CHR-6494 and Structure Activity Relationship was established. An assessment of the inhibitory activity of the lead structures on human Haspin and several other protein kinases is presented. The lead structure was rapidly optimised using a combination of crystal structures and effective docking models, with the best inhibitors exhibiting potent inhibitory activity on Haspin with IC50 between 6 and 100 nM in vitro. The developed inhibitors displayed anti-proliferative properties against various human cancer cell lines in 2D and spheroid cultures and significantly inhibited the migration ability of osteosarcoma U-2 OS cells. Notably, we show that our lead compounds are powerful Haspin inhibitors in human cells, and did not block G2/M cell cycle transition due to improved selectivity against CDK1/CyclinB.

Cell cycle-independent furrowing triggered by phosphomimetic mutations of the INCENP STD motif requires Plk1.

Timely and precise control of Aurora B kinase, the chromosomal passenger complex (CPC) catalytic subunit, is essential for accurate chromosome segregation and cytokinesis. Post-translational modifications of CPC subunits are directly involved in controlling Aurora B activity. Here, we identified a highly conserved acidic STD-rich motif of INCENP that is phosphorylated during mitosis in vivo and by Plk1 in vitro and is involved in controlling Aurora B activity. By using an INCENP conditional-knockout cell line, we show that impairing the phosphorylation status of this region disrupts chromosome congression and induces cytokinesis failure. In contrast, mimicking constitutive phosphorylation not only rescues cytokinesis but also induces ectopic furrows and contractile ring formation in a Plk1- and ROCK1-dependent manner independent of cell cycle and microtubule status. Our experiments identify the phospho-regulation of the INCENP STD motif as a novel mechanism that is key for chromosome alignment and cytokinesis.This article has an associated First Person interview with the first author of the paper.

Correction to: Phenytoin inhibits necroptosis.

The name of the one of the authors was misspelt. The author's surname is Rodriguez, not Rodriquez as originally published. This has been corrected in both the PDF and HTML versions of the Article.

Casein kinase 1ε and 1α as novel players in polycystic kidney disease and mechanistic targets for (R)-roscovitine and (S)-CR8.

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

Phenytoin inhibits necroptosis.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.

Evaluation of the antiprion activity of 6-aminophenanthridines and related heterocycles.

Series of 6-aminophenanthridines and related heterocyclic compounds such as benzonaphtyridines were prepared. Reduction of one of the three aromatic rings was also performed. The compounds were first tested for their antiprion activity in a previously described yeast-based colourimetric prion assay. The most potent derivatives were then assayed ex vivo against the mammalian prion PrP(Sc) in a cell-based assay. Several of the new compounds were found more potent than the parent lead 6-aminophenanthridine. The most promising compounds against yeast and mammalian prions were 8-azido-6-aminophenanthridine (3m), and 7,10-dihydrophenanthridin-6-amine (14). In the mammalian cell-based assay, the IC50 of these two compounds were around 5 µM and 1.8 µM, respectively.

The toll-like receptor agonist imiquimod is active against prions.

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI (+) ] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.

Synthesis of conjugates of 6-aminophenanthridine and guanabenz, two structurally unrelated prion inhibitors, for the determination of their cellular targets by affinity chromatography.

The synthesis of affinity matrices for 6-aminophenanthridine (6AP) and 2,6-dichlorobenzylidenaminoguanidine (Guanabenz, GA), two unrelated prion inhibitors, is described. In both cases, the same simple spacer, epsilon-aminocaproylaminopentanol, was introduced by a Mitsunobu reaction and the choice of the anchoring position of the linker was determined by the study of the residual antiprion activity of the corresponding 6AP or GA conjugates. Very recently, these two affinity matrices were used for chromatography assays leading to the identification of ribosome (via the rRNA) as a common target of these two antiprion drugs. Here, we show, using competition experiments with Quinacrine (QC) and Chlorpromazine (CPZ), two other antiprion drugs, that QC, but not CPZ, may also directly target the rRNA.

Protein folding activity of ribosomal RNA is a selective target of two unrelated antiprion drugs.

BACKGROUND: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. CONCLUSION/SIGNIFICANCE: 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

Antihypertensive drug guanabenz is active in vivo against both yeast and mammalian prions.

BACKGROUND: Prion-based diseases are incurable transmissible neurodegenerative disorders affecting animals and humans. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the discovery of the in vivo antiprion activity of Guanabenz (GA), an agonist of alpha2-adrenergic receptors routinely used in human medicine as an antihypertensive drug. We isolated GA in a screen for drugs active in vivo against two different yeast prions using a previously described yeast-based two steps assay. GA was then shown to promote ovine PrP(Sc) clearance in a cell-based assay. These effects are very specific as evidenced by the lack of activity of some GA analogues that we generated. GA antiprion activity does not involve its agonist activity on alpha2-adrenergic receptors as other chemically close anti-hypertensive agents possessing related mechanism of action were found inactive against prions. Finally, GA showed activity in a transgenic mouse-based in vivo assay for ovine prion propagation, prolonging slightly but significantly the survival of treated animals. CONCLUSION/SIGNIFICANCE: GA thus adds to the short list of compounds active in vivo in animal models for the treatment of prion-based diseases. Because it has been administrated for many years to treat hypertension on a daily basis, without major side-effects, our results suggest that it could be evaluated in human as a potential treatment for prion-based diseases.

Using budding yeast to screen for anti-prion drugs.

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.

alpha1beta1-integrin engagement to distinct laminin-1 domains orchestrates spreading, migration and survival of neural crest cells through independent signaling pathways.

Integrin engagement regulates cell adhesion, shape, migration, growth, and differentiation, but molecular mechanisms coordinating these functions in cells remain unclear. Because of their migratory and differentiation potential, neural crest cells constitute a powerful paradigm to address this question. Here, we describe that laminin-1, a major component of their migration routes, promotes crest cell spreading, migration and survival through two distinct integrin-binding domains that are situated on both sides of its alpha1 subunit and can be separated in the LN-1 elastase proteolytic fragments E1' and E8. Interaction with either domain was mediated by the same integrin alpha1beta1 but produced distinct, complementary responses through specific signaling cascades. FAK activation upon E8 binding induced spreading, formation of actin bundles and focal adhesions, stimulated oriented migration, but failed to support survival. Conversely, Erk activation upon E1' binding promoted long-term survival and random migration without actin reorganization. Consistent with this, interaction with laminin-5 or laminin-10/11, which do not harbor integrin-binding domains in the N-terminal side of their alpha chains, failed to support survival. Thus, the signaling activity and function of integrins might depend on binding domains in their ligands, thereby revealing ligand control of integrin function as a possible mechanism for the modulation and coordination of cell response to adhesive signals.

A yeast-based assay to isolate drugs active against mammalian prions.

Recently, we have developed a yeast-based (Saccharomyces cerevisiae) assay to isolate drugs active against mammalian prions. The initial assumption was that mechanisms controlling prion appearance and/or propagation could be conserved from yeast to human, as it is the case for most of the major cell biology regulatory mechanisms. Indeed, the vast majority of drugs we isolated as active against both [PSI(+)] and [URE3] budding yeast prions turned out to be also active against mammalian prion in three different mammalian cell-based assays. These results strongly argue in favor of common prion controlling mechanisms conserved in eukaryotes, thus validating our yeast-based assay and also the use of budding yeast to identify antiprion compounds and to study the prion world.

Cellular localization and signaling activity of beta-catenin in migrating neural crest cells.

In the vertebrate embryo, development of the neural crest is accompanied by sequential changes in cellular adhesiveness, allowing cells to delaminate from the neural epithelium, to undergo migration through extracellular matrix material, and to coalesce into ganglia of the peripheral nervous system. Because of its dual role in cell adhesion, as a link between cadherins and the actin cytoskeleton, and in cell signaling, as a key mediator of the Wnt-signaling pathway, beta-catenin is a good candidate to play a central role in the control of neural crest cell development. In the present study, we analyzed, by using an in vitro culture system, whether the cellular localization and the signaling activity of beta-catenin are regulated in conjunction with cell migration during ontogeny of trunk neural crest cells in the avian embryo. beta-Catenin molecules were found primarily in association with N-cadherin in the regions of intercellular contacts in most migrating neural crest cells, and only early-migrating cells situated in proximity with the dorsal side of the neural tube showed detectable beta-catenin in their nuclei. This finding indicates that beta-catenin may be recruited for signaling in neural crest cells only transiently at the onset of migration and that sustained beta-catenin signals are not necessary for the progression of migration. The nuclear distribution of beta-catenin within crest cells was not affected upon modification of the N-cadherin-mediated cell-cell contacts, revealing that recruitment of beta-catenin for signaling is not driven by changes in intercellular cohesion during migration. Overstimulation of beta-catenin signals in neural crest cells at the time of their migration, using LiCl treatment or coculture with Wnt-1-producing cells, induced nuclear translocation of beta-catenin and Lef-1 up-regulation in neural crest cells and provoked a marked inhibition of cell delamination and migration. The effect of LiCl and exogenous Wnt-1 on neural crest cells could be essentially attributed to a dramatic decrease in integrin-mediated cell-matrix adhesion as well as a massive reduction of cell proliferation. In addition, although it apparently did not affect expression of neural crest markers, Wnt-1 exposure dramatically affected signaling events involving Notch-Delta, presumably also accounting for the strong reduction in cell delamination. In conclusion, our data indicate that beta-catenin functions primarily in cell adhesion events during migration and may be recruited transiently for signaling during delamination possibly to regulate the balance between cell proliferation and cell differentiation.