Search

Studies confirm Vinca alkaloid, vinflunine as a better inhibitor for stable tubule only polypeptide (STOP) binding to calmodulin than vinblastine. The results help to better understand the superior antitumor efficiency and lower toxicity of vinflunine.

The dimeric 90-kilodalton heat-shock protein undergoes a conformational change in the presence of divalent cations, such as magnesium and calcium, which is due to self-association process. Data do not indicate involvement of the N-terminal fragment, suggesting that C-terminal domain mediates oligomerization.

Research was conducted to prove that the active GTP- and ground GDP-liganded states of tubulin are distinguished by the binding of chiral isomers of ethyl 5-amino-2-methyl-1,2-dihydro-3-phenylpyrido(3,4-b)pyazin-7-yl carbamate. Results indicate that the binding of the R- and S-isomers to the GTP-Mg-tubulin generated a protein conformational change which induced a GTPase activity and the formation of abnormal polymers. Findings also demonstrate that a colchicine-site ligand can induce the polymerization of GDP-tubulin into filamentous structures.

The binding of the R-enantiomer of the antimitotic drug, NSC 370147 ethyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido(3,4-b)pyrazin-7-yl carbamate, to tubulin involves an initial binding followed by a conformational change. The binding kinetics are diphasic and are similar for both tubulin isoforms. The activation energy for the dissociation of the enantiomer from tubulin is larger for the tubulin-S-isomer than the tubulin-R-isomer. The thermodynamic pathway for the S-isomer is unclear.

The ethyl 5-amino-2-methyl-1,2-dihydro-3-phenylpyrido(3,4-b)pyrazin-7-yl carbamate isomers NSC 613863 (R)-(+) and NSC 613862 (S)-(-)(CI980) analogs C179, NSC 337238 and NSC 330770 exhibit differences regarding tubulin binding, polymerization, and GTPase activity. NSC 330770 is a potent inhibitor and binds strongly to tubulin, while C179 interacts weakly with tubulin. NSC 337238 is intermediate between C179 and NSC 330770. Binding of suitable amounts of NSC 330770 and NSC 337238 to tubulin produces anomalous polymers.

A solitary high-affinity binding site on tubulin reveals the binding capacity of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+). The S-isomer reveals an increased level of the affinity constant. Low-affinity binding sites are seen on both enantiomers. The colchicine ring A and C subsites exhibit the binding of both isomers, as seen by competition interactions, revealing the presence of a tryptophan residue. The isomers' variance in fluorescence can be analyzed with another reaction between the enantiomer fluorophore unit and the tubulin site. The two isomers' varying ionization states when bound to tubulin helps to determine the difference in fluorescence.

The binding kinetics of MDL 27048 with tubulin was examined through the use of fluorescence stopped flow techniques. The rate of binding was dependent on the concentration of MDL in pseudo-first-conditions. Analysis of the temperature dependence of displacement experiments for the fast and slow tubulin isoforms determined the activation energy of the conformational change in the dissociation direction. A fast binding analog of the colchicine A-ring hindered the binding of MDL to tubulin while the presence of tropolene methyl ether showed no effect on MDL binding to tubulin. These results implied that MDL shared the A-subsite, but not the C-subsite, with colchine.

The interaction of MDL 27048 with the tubulin-colchine complex was investigated. MDL 27048 exhibited fluorescence characteristics when bound to tubulin and demonstrated various intramolecular rotational relaxation different microenvironment viscosity. The exothermic binding of the antimitotic agent 007 spanned the colchicine and podophyllotoxin binding sites. MDL 27048 and podophyllotoxin showed no effects on either circular dichroism of tubulin or GTPase activity. The dimethoxyphenyl part of MDL 27048 was suggested to bind to the ring A subsite of tubulin while the other part was exposed for other binding with the protein.

Abstract 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 μM 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 × 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.

Abstract 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°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°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.

Abstract Background HIV 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. Results We 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. Conclusions These results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat.

A nuclear magnetic resonance is used to study the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). The presence of an intramolecular disulfide bridge has led to a partial detachment of the C-terminus of Tau and has decreased the overloading of Tau on the MT surface.

A structure-activity relationship of two compounds with potent cytotoxic activity on a CEM leukemia cell line in the context of their binding to tubulin is presented and is compared to combretastatin A-4 and an active compound of the series. Results reveal that although combretastatin A-4 remains the most active compound, the two compounds with potent cytotoxic activity on CEM leukemia cell line have a good antitubulin activity.

Tau is a neuronal microtubule- associated protein that plays a central role in many cellular processes, both physiological and pathological, such as axons stabilization and Alzheimer's disease. A new insight into the mechanisms is gained by which tau binds to tubulin and induces microtubule formation.

Copyright of Canadian Journal of Microbiology is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)