Pascale Barbier

Synthesis and Biological Evaluation of 4-Arylcoumarin Analogues of Combretastatins. Part 2

A series of A-ring variously methoxylated 4-(3-hydroxy-4-methoxyphenyl)coumarins related to combretastatin A-4 was prepared by cross-coupling reactions. Cytotoxicity studies indicated a potent activity against HBL100 cell line. Substitution patterns on A-ring had only a slight effect on antiproliferative activity. For most cytotoxic compounds, the activity as potential modulators of P-gp and BCRP efflux pumps was evaluated. The results show that compounds 2 and 7 were able to restore mitoxantrone accumulation (BCRP) at concentrations similar to that of cyclosporine A. Compound 7 was the most efficient to reverse P-gp activity. All compounds were found to potently inhibit in vitro microtubule formation via a substoichiometric mode of action for the most part. Compounds 1 and 2 were found to have an apparent affinity binding constant similar to that of combretastatin A-4, i.e., 1 × 10 (6) M(-1). The molecular modeling of coumarin derivatives was performed on the basis of the molecular structure of 7, as determined by single-crystal X-ray crystallography. The calculations suggested that the presence of a methoxy group out of the plane of the chromenone moiety is an important steric hindrance factor embedding the accessibility of those molecules inside the binding pocket on tubulin.

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 (T2RB3). 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 T2RB3 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 T2RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T2RB3, T2Stath) 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.

HIV-1 Tat protein enhances Microtubule polymerization

BackgroundHIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.ResultsWe show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria.ConclusionsThese results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat.

Apo-Hsp90 coexists in two open conformational states in solution

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue).

Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules

In Alzheimer disease (AD)‐affected neurons, the Tau protein is found in an aggregated and hyperphosphorylated state. A common hypothesis is that Tau hyperphosphorylation causes its dissociation from the microtubular surface, with consequently a breakdown of the microtubules (MTs) and aggregation of the unbound Tau. We evaluated the effect of Tau phosphorylation on both tubulin assembly and MT binding. We show that the cyclin‐dependent kinase 2/cyclin A3 kinase complex can generate the AT8 and AT180 AD‐specific phospho‐epitopes and use NMR spectroscopy to validate qualitatively and quantitatively the phospho content of our samples. The simultaneous presence of both epitopes disables the tubulin assembly capacity of Tau in conditions whereby Tau is the driving force for the assembly process but does not, however, inhibit MT assembly when the latter is driven by an increased tubulin concentration. When compared to the isolated MT binding repeats (Kd=0.3 µΜ), the phospho‐Tau retains a substantial affinity for preformed MTs (Kd=11 nM), suggesting that the phosphorylated proline‐rich region still participates in the binding event. Our results hence indicate that the sole phosphorylation at the AT8/AT180 epitopes, although leading to a functional defect for Tau, is not sufficient for its dissociation from the MT surface and subsequent aggregation as observed in AD.—Amniai, L., Barbier, P., Sillen, A., Wieruszeski, J.‐M., Peyrot, V., Lippens, G., Landrieu, I. Alzheimer disease specific phosphoepitopes of Tau interfere with assembly of tubulin but not binding to microtubules. FASEB J. 23, 1146–1152 (2009)

Stathmin/Op18 is a novel mediator of vinblastine activity.

MINT‐6603918: tubulin beta (uniprotkb:Q9H4B7), tubulin alpha (uniprotkb:Q71U36) and stathmin (uniprotkb:Q71U36) physically interact (MI:0218) by cosedimentation (MI:0027) MINT‐6603930: tubulin alpha (uniprotkb:Q71U36) physically interacts (MI:0218) with tubulin beta (uniprotkb:Q9H4B7) and stathmin (uniprotkb:P16949) by isothermal titration calorimetry (MI:0065)

Tau aggregation in Alzheimer's disease: what role for phosphorylation?

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper-phosphorylated but non-aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly-anions.

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

Summary 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 &agr;-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 &agr;-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.

Mechanism of Tau-Promoted Microtubule Assembly As Probed by NMR Spectroscopy

Determining the molecular mechanism of the neuronal Tau protein in the tubulin heterodimer assembly has been a challenge owing to the dynamic character of the complex and the large size of microtubules. We use here defined constructs comprising one or two tubulin heterodimers to characterize their association with a functional fragment of Tau, named TauF4. TauF4 binds with high affinities to the tubulin heterodimer complexes, but NMR spectroscopy shows that it remains highly dynamic, partly because of the interaction with the acidic C-terminal tails of the tubulin monomers. When bound to a single tubulin heterodimer, TauF4 is characterized by an overhanging peptide corresponding to the first of the four microtubule binding repeats of Tau. This peptide becomes immobilized in the complex with two longitudinally associated tubulin heterodimers. The longitudinal associations are favored by the fragment and contribute to Taus functional role in microtubule assembly.

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

Tau antagonizes tracking of end-binding proteins (EBs) at microtubule ends, a process requiring the C-terminal part of EBs and the microtubule-binding sites of tau. The inhibiting activity of tau on EB properties is regulated by tau phosphorylation. The interplay between EBs and tau proteins results in modulation of microtubule dynamics.