2-Aminothiazole-Flavonoid Hybrid Derivatives Binding to Tau Protein and Responsible for Antitumor Activity in Glioblastoma.

Tau protein has been described for several decades as a promoter of tubulin assembly into microtubules. Dysregulation or alterations in Tau expression have been related to various brain cancers, including the highly aggressive and lethal brain tumor glioblastoma multiform (GBM). In this respect, Tau holds significant promise as a target for the development of novel therapies. Here, we examined the structure-activity relationship of a new series of seventeen 2-aminothiazole-fused to flavonoid hybrid compounds (TZF) on Tau binding, Tau fibrillation, and cellular effects on Tau-expressing cancer cells. By spectrofluorometric approach, we found that two compounds, 2 and 9, demonstrated high affinity for Tau and exhibited a strong propensity to inhibit Tau fibrillation. Then, the biological activity of these compounds was evaluated on several Tau-expressing cells derived from glioblastoma. The two lead compounds displayed a high anti-metabolic activity on cells related to an increased fission of the mitochondria network. Moreover, we showed that both compounds induced microtubule bundling within newly formed neurite-like protrusions, as well as with defection of cell migration. Taken together, our results provide a strong experimental basis to develop new potent molecules targeting Tau-expressing cancer cells, such as GBM.

Tau Protein as Therapeutic Target for Cancer? Focus on Glioblastoma.

Despite being extensively studied for several decades, the microtubule-associated protein Tau has not finished revealing its secrets. For long, Tau has been known for its ability to promote microtubule assembly. A less known feature of Tau is its capability to bind to cancer-related protein kinases, suggesting a possible role of Tau in modulating microtubule-independent cellular pathways that are associated with oncogenesis. With the intention of finding new therapeutic targets for cancer, it appears essential to examine the interaction of Tau with these kinases and their consequences. This review aims at collecting the literature data supporting the relationship between Tau and cancer with a particular focus on glioblastoma tumors in which the pathological significance of Tau remains largely unexplored. We will first treat this subject from a mechanistic point of view showing the pivotal role of Tau in oncogenic processes. Then, we will discuss the involvement of Tau in dysregulating critical pathways in glioblastoma. Finally, we will outline promising strategies to target Tau protein for the therapy of glioblastoma.

Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway.

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau.

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation.

Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges.

Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αß-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αß-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αß-tubulin.

Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents.

The synthesis of twenty-six 4-arylcoumarin analogues of combretastatin A-4 (CA-4) led to the identification of two new compounds (25 and 26) with strong cytotoxic activity. Both compounds had a high cytotoxic effect on a CA-4-resistant colon adenocarcinoma cell line (HT29D4). The compounds affected cell cycle progression characterized by a mitotic block. The activity of these compounds against microtubules both in vitro and in cells was examined and both compounds were found to potently inhibit in vitro microtubule formation via a sub-stoichiometric mode like CA-4. By immunofluorescence, it was observed that both compounds induced strong microtubule network disruption. Our results provide a strong experimental basis to develop new potent anti-tubulin molecules targeting CA-4-resistant cancer cells.

Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells.

Microtubules (MTs) play an important role in many cellular processes and are dynamic structures regulated by an important network of microtubules-associated proteins, MAPs, such as Tau. Tau has been discovered as an essential factor for MTs formation in vitro, and its region implicated in binding to MTs has been identified. By contrast, the affinity, the stoichiometry, and the topology of Tau-MTs interaction remain controversial. Indeed, depending on the experiment conditions a wide range of values have been obtained. In this chapter, we focus on three biophysical methods, turbidimetry, cosedimentation assay, and Förster Resonance Energy Transfer to study Tau-tubulin interaction both in vitro and in cell. We highlight precautions that must be taken in order to avoid pitfalls and we detail the nature of the conclusions that can be drawn from these methods about Tau-tubulin interaction.

Peptide screen identifies a new NADPH oxidase inhibitor: impact on cell migration and invasion.

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.

Interactions of long-chain homologues of colchicine with tubulin.

Several colchicine analogues in which the N-acetyl residue has been replaced by aliphatic, straight-chain acyl moieties, have been synthesized. These compounds show high cytotoxic activity at the nanomolar level against the tumoral cell lines HT-29, MCF-7 and A549. Some of them exhibit activities in the picomolar range against the HT-29 line and are thus two to three orders of magnitude more cytotoxic than colchicine. In this specific cell line, the activities were found to be closely related to the length of the acyl carbon chain, an increase in the latter giving rise to an increase in the cytotoxicity with a maximum in the range of 10-12 carbon atoms, followed by a decrease in activity with still longer chains. Some of the compounds inhibit microtubule assembly and induce the formation of abnormal polymers and present in most cases better apparent affinity constants than colchicine. In addition, at IC50 concentrations the analogues block the cell cycle of A549 cells in the G2/M phase. Molecular docking studies suggest that, while interactions of the colchicine analogues with the colchicine binding site at ß-tubulin are still present, the increase in the acyl chain length leads to the progressive development of new interactions, not present in colchicine itself, with the neighboring α-tubulin subunit. Indeed, sufficiently long acyl chains span the intradimer interface and contact with a hydrophobic groove in α-tubulin. It is worth noting that some of the compounds show cytotoxicity at concentrations three orders of magnitude lower than colchicine. Their pharmacological use in cancer therapy could possibly be performed with lower dosages and be thus endowed with less acute toxicity problems than in the case of colchicine.

Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism.

Proper regulation of microtubule dynamics is essential for cell functions and involves various microtubule-associated proteins (MAPs). Among them, end-binding proteins (EBs) accumulate at microtubule plus ends, whereas structural MAPs bind along the microtubule lattice. Recent data indicate that the structural MAP tau modulates EB subcellular localization in neurons. However, the molecular determinants of EB/tau interaction remain unknown, as is the effect of this interplay on microtubule dynamics. Here we investigate the mechanisms governing EB/tau interaction in cell-free systems and cellular models. We find that tau inhibits EB tracking at microtubule ends. Tau and EBs form a complex via the C-terminal region of EBs and the microtubule-binding sites of tau. These two domains are required for the inhibitory activity of tau on EB localization to microtubule ends. Moreover, the phosphomimetic mutation S262E within tau microtubule-binding sites impairs EB/tau interaction and prevents the inhibitory effect of tau on EB comets. We further show that microtubule dynamic parameters vary, depending on the combined activities of EBs and tau proteins. Overall our results demonstrate that tau directly antagonizes EB function through a phosphorylation-dependent mechanism. This study highlights a novel role for tau in EB regulation, which might be impaired in neurodegenerative disorders.

Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

Stathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies have strongly suggested that stathmin could act by sequestering tubulin and/or by binding to MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. By using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analyzed the ability of stathmin and its phosphorylated forms (on Ser16, -25, -38, and -63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin-tubulin interactions at the MT plus ends and in the cytosol. Of interest, we also observed a novel pool of stathmin bound along the MT. Expression of truncated stathmin and use of MT-stabilizing taxol further showed that the C-terminal domain of stathmin is the main contributor to this binding and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus end of a depolymerizing MT reaches stathmin molecules.-Nouar, R., Breuzard, G., Bastonero, S., Gorokhova, S., Barbier, P., Devred, F., Kovacic, H., Peyrot, V. Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

Sequential binding of calcium ions to the B-repeat domain of SdrD from Staphylococcus aureus.

Biofilms of live bacteria forming on medical devices and implants contribute significantly to bacterial blood dissemination and to the spread of nosocomial infections. Cell surface SdrD protein plays a key role in the attachment of Staphylococcus aureus to the extracellular matrix (ECM) and in the formation of biofilm. SdrD binds calcium ions using its B1-B5 region bearing EF-hand Ca-binding sites, leading to conformational changes in the structure of SdrD. This alters the distance between the bacterial surface and the ECM-interacting domain of SdrD in a spring-like fashion, participating in bacterial attachment. In this study we investigated calcium binding to EF-hand sites of SdrD using isothermal titration calorimetry and determined the impact of this process on SdrD's thermodynamic stability. This allowed us to propose a model of B1-B5 reorganization upon binding of calcium and to get new insight into the molecular mechanism of SdrD's action.

Stathmin potentiates vinflunine and inhibits Paclitaxel activity.

Cell biology and crystallographic studies have suggested a functional link between stathmin and microtubule targeting agents (MTAs). In a previous study we showed that stathmin increases vinblastine (VLB) binding to tubulin, and that conversely VLB increases stathmin binding to tubulin. This constituted the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and revealed a new mechanism of action for VLB. The question remained if the observed interaction was specific for this drug or represented a general phenomenon for all MTAs. In the present study we investigated the binding of recombinant stathmin to purified tubulin in the presence of paclitaxel or another Vinca alkaloid, vinflunine, using Isothermal Titration Calorimetry (ITC). These experiments revealed that stathmin binding to tubulin is increased in the presence of vinflunine, whereas no signal is observed in the presence of paclitaxel. Further investigation using turbidity and co-sedimentation showed that stathmin inhibited paclitaxel microtubule-stabilizing activity. Taken together with the previous study using vinblastine, our results suggest that stathmin can be seen as a modulator of MTA activity and binding to tubulin, providing molecular explanation for multiple previous cellular and in vivo studies showing that stathmin expression level affects MTAs efficiency.

Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.

Quantitative analysis of tau-microtubule interaction using FRET.

The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αß-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and ß-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.

The indolylcoumarin COUFIN exhibits potent activity against renal carcinoma cells without affecting hematopoietic system.

The present work describes the anticancer activity of a new indolylcoumarin named COUFIN and more specifically, its efficiency against clear cell renal carcinoma (CCRC). COUFIN inhibited microtubule formation and bound on tubulin to or near the colchicine site. In vitro, COUFIN showed potent anticancer activity on renal carcinoma cells (RCC) both in monolayer (2D culture) (IC50 of 88 ± 8 nM) and multicellular tumor spheroid (3D culture) (IC50 of 180 ± 20 nM). The compound blocked cell cycle transition at G2/M phase, induced a subsequent apoptotic process but did not modulate clonal growth of CFU-GM. On the other hand, the coumarin derivative decreased the activity of P-gp and BCRP but was not substrate for these ABC pumps. In vivo, the indolylcoumarin increased the survival rate after 3 weeks of treatment. Based on the present study, COUFIN was identified as a bifunctional molecule able to inhibit renal carcinoma cells proliferation without being effluxed by ABC proteins. Thus COUFIN could be a promising chemotherapeutic agent for treating tumor cells over-expressing efflux pumps and tumor cells irrigated by vessels lined with endothelial cells responsible of poor distribution of conventional anticancer agents.

Molecular mechanisms of Tau binding to microtubules and its role in microtubule dynamics in live cells.

Despite extensive studies, the molecular mechanisms of Tau binding to microtubules (MTs) and its consequences on MT stability still remain unclear. It is especially true in cells where the spatiotemporal distribution of Tau-MT interactions is unknown. Using Förster resonance energy transfer (FRET), we showed that the Tau-MT interaction was distributed along MTs in periodic hotspots of high and low FRET intensities. Fluorescence recovery after photobleaching (FRAP) revealed a two-phase exchange of Tau with MTs as a rapid diffusion followed by a slower binding phase. A real-time FRET assay showed that high FRET occurred simultaneously with rescue and pause transitions at MT ends. To further explore the functional interaction of Tau with MTs, the binding of paclitaxel (PTX), tubulin acetylation induced by trichostatin A (TSA), and the expression of non-acetylatable tubulin were used. With PTX and TSA, FRAP curves best fitted a single phase with a long time constant, whereas with non-acetylatable α-tubulin, curves best fitted a two phase recovery. Upon incubation with PTX and TSA, the number of high and low FRET hotspots decreased by up to 50% and no hotspot was observed during rescue and pause transitions. In the presence of non-acetylatable α-tubulin, a 34% increase in low FRET hotspots occurred, and our real-time FRET assay revealed that low FRET hotspots appeared with MTs recovering growth. In conclusion, we have identified, by FRET and FRAP, a discrete Tau-MT interaction, in which Tau could induce conformational changes of MTs, favoring recovery of MT self-assembly.

FRET and FRAP imaging: approaches to characterise tau and stathmin interactions with microtubules in cells.

Microtubules (MTs) are involved in many crucial processes such as cell morphogenesis, mitosis and motility. These dynamic structures resulting from the complex assembly of tubulin are tightly regulated by stabilising MT-associated proteins (MAPs) such as tau and destabilising proteins, notably stathmin. Because of their key role, these MAPs and their interactions have been extensively studied using biochemical and biophysical approaches, particularly in vitro. Nevertheless, numerous questions remain unanswered and the mechanisms of interaction between MT and these proteins are still unclear in cells. Techniques coupling cell imaging and fluorescence methods, such as Förster resonance energy transfer and fluorescence recovery after photobleaching, are excellent tools to study these interactions in situ. After describing these methods, we will present emblematic data from the literature and unpublished experimental results from our laboratory concerning the interactions between MTs, tau and stathmin in cells.

Synthesis and biological evaluation of novel anticancer bivalent colchicine-tubulizine hybrids.

A series of novel antimitotic hybrids were synthesized in good yields by linking of azide-containing colchicine congeners with acetylene-substituted tubulizine-type derivatives using copper-mediated 1,3-dipolar cycloaddition. Obtained compounds exhibit good cytotoxicity against HBL100 epithelial cell lines (IC(50)=0.599-2.93 µÐœ). Several newly synthesized compounds are the substoichiometric inhibitors of microtubule assembly (R=0.41-0.78). The results highlight the importance of the length of spacer linking the tubulin binding ligands in heterodimeric molecules.

New insights into tau-microtubules interaction revealed by isothermal titration calorimetry.

Microtubule dynamic instability is tightly regulated by coordinated action of stabilizing and destabilizing microtubule associated proteins. Among the stabilizing proteins, tau plays a pivotal role in both physiological and pathological processes. Nevertheless, the detailed mechanism of tau-tubulin interaction is still subject to controversy. In this report, we studied for the first time tau binding to tubulin by a direct thermodynamic method in the absence of any tubulin polymerization cofactors that could influence this process. Isothermal titration calorimetry enabled us to evidence two types of tau-tubulin binding modes: one corresponding to a high affinity binding site with a tau:tubulin stoichiometry of 0.2 and the other one to a low affinity binding site with a stoichiometry of 0.8. The same stoichiometries were obtained at all temperatures tested (10-37°C), indicating that the mechanism of interaction does not depend on the type of tubulin polymer triggered upon tau binding. These findings allowed us to get new insights into the topology of tau on microtubules.