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.

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.

Hsp90 oligomerization process: How can p23 drive the chaperone machineries?

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process.

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.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0 nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

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.

Binding of two zinc ions promotes liquid-liquid phase separation of Tau.

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc.

Identification of the three zinc-binding sites on tau protein.

Tau protein has been extensively studied due to its key roles in microtubular cytoskeleton regulation and in the formation of aggregates found in some neurodegenerative diseases. Recently it has been shown that zinc is able to induce tau aggregation by interacting with several binding sites. However, the precise location of these sites and the molecular mechanism of zinc-induced aggregation remain unknown. Here we used Nuclear Magnetic Resonance (NMR) to identify zinc binding sites on tau. These experiments revealed three distinct zinc binding sites on tau, located in the N-terminal part, the repeat region and the C-terminal part. Further analysis enabled us to show that the N-terminal and the C-terminal sites are independent of each other. Using molecular simulations, we proposed a model of each site in a complex with zinc. Given the clinical importance of zinc in tau aggregation, our findings pave the way for designing potential therapies for tauopathies.

Biochemical and biophysical characterization of the Mg2+-induced 90-kDa heat shock protein oligomers.

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

Stathmin and interfacial microtubule inhibitors recognize a naturally curved conformation of tubulin dimers.

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.

Allocolchicinoids bearing a Michael acceptor fragment for possible irreversible binding of tubulin.

We describe an attempt to apply the concept of covalent binding towards the highly active allocolchicinoids selected on the basis of SAR analysis of previously synthesized molecules. To achieve the irreversible binding of the agent to the cysteine residues of the colchicine site of tubulin protein, we synthesized a number of new allocolchicinoids bearing the acceptor moiety. Some of the new derivatives possess cytotoxic activity against COLO-357, BxPC-3, HaCaT, and HEK293 cell lines in a low nanomolar range of concentrations. A substoichiometric mode of microtubule assembly inhibition was demonstrated. The most active compounds possess close to colchicine general toxicity on mice.

Discovery of dihydrofuranoallocolchicinoids - Highly potent antimitotic agents with low acute toxicity.

Two series of heterocyclic colchicinoids bearing ß-methylenedihydrofuran or 2H-pyran-2-one fragments were synthesized by the intramolecular Heck reaction. Methylenedihydrofuran compounds 9a and 9h were found to be the most cytotoxic among currently known colchicinoids, exhibiting outstanding antiproliferative activity on tumor cell lines in picomolar (0.01-2.1 nM) range of concentrations. Compound 9a potently and substoichiometrically inhibits microtubule formation in vitro, being an order of magnitude more active in this assay than colchicine. Derivatives 9a and 9h revealed relatively low acute toxicity in mice (LD50 ≥ 10 mg/kg i.v.). The X-Ray structure of colchicinoid 9a bound to tubulin confirmed interaction of this compound with the colchicine binding site of tubulin.

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models.

BACKGROUND: Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect. METHODS: The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours. RESULTS: In the four human and the murine glioblastoma cell lines tested, 10 muM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 x 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB. CONCLUSION: These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model.

Caulerpenyne binding to tubulin: structural modifications by a non conventional pharmacological agent.

The most widely used molecules in cancer chemotherapy are Vinca-alkaloids and Taxoids, numerous chemists attempted the synthesis of analogs which bind to their well-known tubulin pharmacological site. Unfortunately, tumors develop resistance to these compounds; therefore the definition of anchoring points and potential binding sites for new drugs on tubulin is of major interest. Caulerpenyne (Cyn), the major secondary metabolite synthesized by the green marine alga Caulerpa taxifolia could be one of these drugs, since it inhibits the assembly of tubulin and MTP (Barbier et al., 2001). We observed that the tubulin-Cyn complex is poorly reversed. Cyn did not bind to sulfhydryl groups and the measure of the extent of binding is 1.6 +/- 0.2 suggesting two potential binding sites. Then, we demonstrated by competition measurements that Cyn did not interact to colchicine, Taxol and Vinca-alkaloid binding domain. Finally, mass spectrometric analysis of proteolytic cleavage of tubulin-Cyn complex demonstrated that Cyn did not bind covalently to tubulin and evidenced two good candidate regions for Cyn binding, one on alpha-tubulin and the other on beta-tubulin.

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat.

BACKGROUND: During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. RESULTS: To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20 degrees C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37 degrees C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. CONCLUSION: Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

Stathmin/Op18 is a novel mediator of vinblastine activity.

Microtubule (MT) dynamic instability is tightly regulated by stabilizing and destabilizing proteins, the latter being exemplified by stathmin/Op18, a protein known to destabilize MTs. Studies in cells have indicated that the level of stathmin expression modifies the cytotoxicity of antimicrotubule drugs, such as vinblastine (VLB). Using isothermal titration calorimetry and analytical ultracentrifugation, we show that VLB increases the affinity of stathmin for tubulin 50-fold (and vice versa). These results are the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and reveal a new mechanism of action for VLB.

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.

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.

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.