Methods for Studying Vinca Alkaloid Interactions With Tubulin

Lobert, Sharon; Correia, John J.

doi:10.1007/978-1-59745-442-1_18

Cited by 3 publications

(9 citation statements)

References 35 publications

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“…The technique was also particularly successful in the study of the binding of the vinca alkaloids (vinblastine, vincristine, catharanthine) to tubulin (Na and Timasheff, 1980a,b;Na and Timasheff, 1986a,b). It was further developed in Correia's team, who analyzed thermodynamic data from many vinca alcaloid and analogues: from classical family members such as vinorelbine and vinflunine to new compounds such as eribulin (Alday and Correia, 2009;Chatterjee et al, 2001;Lobert and Correia, 2007;Lobert et al, 1998).…”

Section: Rationalementioning

confidence: 99%

Microtubule and MAPs

Devred¹,

Barbier²,

Lafitte³

et al. 2010

Methods in Cell Biology

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Microtubules are implicated in many essential cellular processes such as architecture, cell division, and intracellular traffic, due to their dynamic instability. This dynamicity is tightly regulated by microtubule-associated proteins (MAPs), such as tau and stathmin. Despite extensive studies motivated by their central role in physiological functions and pathological role in neurodegenerative diseases and cancer, the precise mechanisms of tau and stathmin binding to tubulin and their consequences on microtubule stability are still not fully understood. One of the most crucial points missing is a quantitative thermodynamic description of their interaction with tubulin/microtubules and of the tubulin complexes formed upon these interactions. In this chapter, we will focus on the use of analytical ultracentrifugation, isothermal titration calorimetry, and nuclear magnetic resonance-three powerful and complementary techniques in the field of MAP-tubulin/microtubule interactions, in addition to the spectrometric techniques and co-sedimentation approach. We will present the limits of these techniques to study this particular interaction and precautions that need to be taken during MAPs preparation. Understanding the molecular mechanisms that govern MAPs action on microtubular network will not only shed new light on the role of this crucial family of protein in the biology of the cell, but also hopefully open new paths to increase the therapeutic efficiency of microtubule-targeting drugs in cancers therapies and neurodegeneratives diseases prevention.

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Section: Rationalementioning

confidence: 99%

Microtubule and MAPs

Devred¹,

Barbier²,

Lafitte³

et al. 2010

Methods in Cell Biology

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“…The vinca alkaloid class of antimitotics (including the clinically useful drugs vinblastine, vincristine, vinorelbine, and vinflunine, as well as dolastatin 10) bind at the interdimer interface (8) and cause microtubule depolymerization while inducing spiral and ringlike polymer formation. Halichondrin B and analogues are thought to compete for vinblastine binding and in general have been thought to act by a mode of action similar, although not identical, to that of vinca alkaloids (9-15).The in vitro techniques used to ascertain the mode of action of antimitotic drugs include turbidity, electron microscopy, and analytical ultracentrifugation (16,17). The advantage of analytical ultracentrifugation (AUC) 1 is the ability to extract the mode of molecular interaction, the stoichiometry, and the energetics of the process, i.e., the equilibrium constants for the molecular mechanism (1, 16-20).…”

mentioning

confidence: 99%

“…The advantage of analytical ultracentrifugation (AUC) 1 is the ability to extract the mode of molecular interaction, the stoichiometry, and the energetics of the process, i.e., the equilibrium constants for the molecular mechanism (1,(16)(17)(18)(19)(20). Here we investigate the interaction of two halichondrin B analogues, eribulin (previously ER-086526, E7389) and ER-076349 (13), with tubulin by quantitative AUC methods.…”

mentioning

confidence: 99%

Macromolecular Interaction of Halichondrin B Analogues Eribulin (E7389) and ER-076349 with Tubulin by Analytical Ultracentrifugation

Alday

Correia

2009

Biochemistry

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Halichondrin B is an antimitotic drug that inhibits microtubule assembly. To understand the molecular details of its interaction with tubulin, we investigated the binding of two halichondrin B analogues, eribulin (previously, ER-086526, E7389) and ER-076349, to tubulin by quantitative analytical ultracentrifugation. Eribulin is currently undergoing phase III clinical trials for cancer; ER-076349 is a closely related analogue with C.35 hydroxyl instead of C.35 primary amine [Towle, M. J., et al. (2001) Cancer Res. 61, 1013]. Below the critical concentration for microtubule assembly and in the presence of GDP, tubulin undergoes weak self-association into short curved oligomers. Eribulin inhibits this oligomer formation 4-6-fold, while ER-076349 slightly stimulates oligomer formation by 2-fold. This is in contrast to vinblastine which strongly stimulates large spiral polymers by 1000-fold under these same conditions. Vinblastine-induced spiral formation is strongly inhibited by both eribulin and ER-076349. Colchicine binding to the intradimer interface has no significant effect on small oligomer formation or the inhibitory activity of eribulin on this process. These results suggest that halichondrin B analogues bind to the interdimer interface or to the beta-subunit alone, disrupt polymer stability, and compete with vinblastine-induced spiral formation. Stathmin is known to form a tight 1:2 complex with tubulin. Eribulin strongly inhibits formation of the 1:2 stathmin-tubulin complex (>3.3 kcal/mol), while ER-076349 weakens formation of the 1:2 complex by approximately 1.9 kcal/mol. These results suggest that eribulin is a global inhibitor of tubulin polymer formation, disrupting tubulin-tubulin contacts at the interdimer interface. ER-076349 also perturbs tubulin-tubulin contacts, but in a more polymer specific manner, reflecting adaptability of the interdimer interface to drug and polymer polymorphism. These results suggest halichondrin B analogues exhibit unique tubulin-based activities that may underlie the clinical utility of these compounds.

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“…Goat antimouse IgG/Alexa-Fluor 488 conjugate was diluted 1:200 in 10% goat serum and incubated with cells for 1 h, then washed three times with PBST. Cells were incubated in PBS containing 10 μg/mL propidium iodide and 1 μg/mL RNase A for 15 min, washed twice with PBS, once with H 2 O, and mounted onto microscope slides using 8 μL of Vectashield mounting medium and sealed with colorless nail polish . Samples were visualized immediately on a Zeiss LSM 510 laser scanning confocal microscope, 63× oil DIC objective, 1.4 NA.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were incubated in PBS containing 10 μg/mL propidium iodide and 1 μg/mL RNase A for 15 min, washed twice with PBS, once with H 2 O, and mounted onto microscope slides using 8 μL of Vectashield mounting medium and sealed with colorless nail polish. 45 Samples were visualized immediately on a Zeiss LSM 510 laser scanning confocal microscope, 63Â oil DIC objective, 1.4 NA. Wide-field images were acquired by moving the stage to 10-15 random locations on each slide, thereafter only adjusting the stage in the z-direction to bring a maximal number of cells into focus.…”

Section: Methodsmentioning

confidence: 99%

Phenylcinnamides as Novel Antimitotic Agents

et al. 2010

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Compound 8H is a phenylcinnamide that induces G2/M-phase cell cycle arrest and cell death in cancer cell lines. Here we show that 8H exerts its cytotoxic activity through disruption of microtubule dynamics in vitro and in cell culture. A series of cinnamide derivatives were synthesized and evaluated, and several new compounds were identified that improve on the activity of the parent compound, with IC(50) values for induction of cell death ranging from 1 to 10 microM. Notably, these compounds retain potency in the HL-60/VCR leukemia cell line, which is resistant to antimitotic cancer drugs vincrisitine and paclitaxel through up-regulation of P-glycoprotein drug efflux pumps. As P-glycoprotein expression is often responsible for drug resistance in cancer and the exclusion of compounds from the central nervous system, 8H and its derivatives merit further examination as potential antimitotic therapeutics, specifically for brain cancers and cancers that are resistant to standard antimitotic agents.

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Methods for Studying Vinca Alkaloid Interactions With Tubulin

Cited by 3 publications

References 35 publications

Microtubule and MAPs

Microtubule and MAPs

Macromolecular Interaction of Halichondrin B Analogues Eribulin (E7389) and ER-076349 with Tubulin by Analytical Ultracentrifugation

Phenylcinnamides as Novel Antimitotic Agents

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