<i>N</i>‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yI)colcemid, a probe for different classes of colchincine‐binding site on tubulin

Sengupta, S. K.; Puri, Kamal D.; Surolia, Avadhesha; Roy, Siddhartha; Bhattacharyya, Bhabatarak

doi:10.1111/j.1432-1033.1993.tb17673.x

Cited by 9 publications

(5 citation statements)

References 23 publications

(4 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The time dependence of NBD-isocolcemid binding was tested by observing its fluorescence as a function of time after mixing 3 µM tubulin with 5 µM NBD-isocolcemid at 37 °C. The profile of fluorescence increase is (Figure 2A) similar to that for the NBD-colcemid-tubulin interaction reported from this laboratory (17). It is characterized by a very rapid phase which is completed within the time of mixing of the protein and the drug, and a slow phase like the type which is observed for the colchicine binding to tubulin.…”

Section: Resultssupporting

confidence: 77%

“…The values of k 1 and k -1 thus obtained are (7.37 ( 0.70) × 10 5 M -1 s -1 and 7.82 ( 2.74 s -1 , respectively. These values are similar to that obtained for the tubulin-NBD-colcemid interaction (17). The value of the dissociation constant could not be directly determined due to the lack of the displacing ligand, as neither colchicine nor its derivatives were able to displace NBD-isocolcemid bound to tubulin at the fast site.…”

Section: Competitive Inhibition Of [ 3 H]colchicine Binding To Tubuli...supporting

confidence: 79%

See 1 more Smart Citation

NBD-Isocolcemid−Tubulin Interaction: A Novel One-Step Reaction Involving No Conformational Adjustment of Reactants

et al. 2000

Self Cite

View full text Add to dashboard Cite

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

show abstract

Section: Resultssupporting

confidence: 77%

Section: Competitive Inhibition Of [ 3 H]colchicine Binding To Tubuli...supporting

confidence: 79%

NBD-Isocolcemid−Tubulin Interaction: A Novel One-Step Reaction Involving No Conformational Adjustment of Reactants

et al. 2000

Self Cite

View full text Add to dashboard Cite

show abstract

“…70 Thus, it seems quite natural that an interchange of the >CO and -OCH 3 groups as in isocolchicine would affect these hydrogen bond formation in addition to steric constraints. 71 These two drugs as seen from models have different orientation of their colchicine moiety, while the dansyl moieties occupy almost the same position. Moreover, the conformations of a-tubulin are not significantly altered in these complexes (rmsd < 0.1Å ), but large deviations in the respective b subunits are noticeable (rmsd $0.9 Å ).…”

Section: D E S I G N O F a C T I V E I S O C O L C H I C I N E A mentioning

confidence: 99%

“…69 On the contrary, the binding of NBD-colcemid (4q) to tubulin is a two-step process like colchicine. 71 NH-dansylcolchicine (4r), another B-ring analog of colchicine acts like NBD-colcemid (4q) and binds tubulin in a two-step process whereas binding of its iso-analog to tubulin occurs in one step. 70 For this drug, the kinetics is manifested with the conventional linear dependence of the observed rate constant on drug concentration with its parent compound, isocolchicine.…”

Section: D E S I G N O F a C T I V E I S O C O L C H I C I N E A mentioning

confidence: 99%

Anti‐mitotic activity of colchicine and the structural basis for its interaction with tubulin

Bhattacharyya

Panda

Gupta

et al. 2007

Medicinal Research Reviews

Self Cite

434

311

View full text Add to dashboard Cite

In this review, an attempt has been made to throw light on the mechanism of action of colchicine and its different analogs as anti-cancer agents. Colchicine interacts with tubulin and perturbs the assembly dynamics of microtubules. Though its use has been limited because of its toxicity, colchicine can still be used as a lead compound for the generation of potent anti-cancer drugs. Colchicine binds to tubulin in a poorly reversible manner with high activation energy. The binding interaction is favored entropically. In contrast, binding of its simple analogs AC or DAAC is enthalpically favored and commences with comparatively low activation energy. Colchicine-tubulin interaction, which is normally pH dependent, has been found to be independent of pH in the presence of microtubule-associated proteins, salts or upon cleavage of carboxy termini of tubulin. Biphasic kinetics of colchicines-tubulin interaction has been explained in light of the variation in the residues around the drug-binding site on beta-tubulin. Using the crystal structure of the tubulin-DAMAcolchicine complex, a detailed discussion on the pharmacophore concept that explains the variation of affinity for different colchicine site inhibitors (CSI) has been discussed.

show abstract

“…These properties indicate FC is of low utility for long term microscopy studies. Both dansyllabeled colchicine (DC)9 and NBD-labeled colcemid (NBC)10,11 have been reported. Unfortunately both DC and NBC have low quantum yields and are highly environmentally sensitive.…”

mentioning

confidence: 99%

Synthesis and characterization of BODIPY-labeled colchicine

Arnold

Ranaivo

Guy

2008

Bioorganic & Medicinal Chemistry Letters

View full text Add to dashboard Cite

Two BODIPY-labeled colchicine derivatives were synthesized and shown to bind to tubulin but only partially inhibit tubulin polymerization in the presence of GTP. Cytotoxicity studies were carried out in HeLa, HepG2, Raji and Vero cells. Apoptosis-inducing properties were determined by caspase 3/7 activity and flow cytometry and interactions between the derivatives and tubulin were verified by fluorescence microscopy of living cells.Colchicine, a natural product extracted from Colchicum autumnale, is used, despite serious side effects, as treatment of gout ( Figure 1). Colchicine can bind to tubulin, 1,2 initiating a conformational change of tubulin subunits that leads to rapid depolymerization of microtubules. 3 In contrast, paclitaxel stabilizes the α,β-tubulin dimer by occupying a different binding site than colchicine. 4We desired to take advantage of the differential binding of colchicine to un-polymerized tubulin in order to track sub-cellular localization of tubulin dimers and small oligimers in living, nonpermeablized cells. We were particularly interested in the flux of tubulin in neurons and the flagella of Chlamydomona; and in understanding the role of sequestration in normal function of tubulin structures in these cells.Meeting the project goals requires a colchicine analog that is cell permeable, non-cytotoxic, tubulin binding, stable for extended microscopy studies (non-bleaching), excited at wavelengths compatible with living cells (green to red, not blue), of good quantum yield (to detect low abundance dimers), insensitive to environment, and minimally inhibiting of tubulin polymerization. This is a demanding set of requirements.Prior approaches have used anti-tubulin antibodies or fluorescein-labeled colchicine (FC) to study the localization of tubulin in fixed cells. 5-7 Unfortunately, spectrofluorometry experiments indicated that the anionic nature of FC resulted in binding properties different from those of unlabeled colchicine. 8 Additionally, FC is sensitive to photo-bleaching, is highly pH sensitive, and has limited cellular permeability. These properties indicate FC is of low utility for long term microscopy studies. Both dansyllabeled colchicine (DC) 9 and NBDlabeled colcemid (NBC) 10,11 have been reported. Unfortunately both DC and NBC have low quantum yields and are highly environmentally sensitive. Additionally, DC has an excitation wavelength in the deep blue region. Together these properties render these two reagents of low Correspondence to: R. Kiplin Guy. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Herein, we report the synthesis...

show abstract

N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yI)colcemid, a probe for different classes of colchincine‐binding site on tubulin

Cited by 9 publications

References 23 publications

NBD-Isocolcemid−Tubulin Interaction: A Novel One-Step Reaction Involving No Conformational Adjustment of Reactants

NBD-Isocolcemid−Tubulin Interaction: A Novel One-Step Reaction Involving No Conformational Adjustment of Reactants

Anti‐mitotic activity of colchicine and the structural basis for its interaction with tubulin

Synthesis and characterization of BODIPY-labeled colchicine

Contact Info

Product

Resources

About