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

Sengupta, S. K.; Banerjee, Sagarika; Chakrabarti, Gopal; Mahapatra, Pradip Kumar; Roy, Siddhartha; Bhattacharyya, Bhabatarak

doi:10.1021/bi991530e

Cited by 5 publications

(8 citation statements)

References 27 publications

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“…Then all samples were analyzed using SDS-PAGE (12% polyacrylamide gel) followed by Coomassie blue staining. 45 Isothermal Titration Microcalorimetry (ITC). ITC measurements were performed on a VP-ITC calorimeter (Microcal Inc., Northampton, MA).…”

Section: Methodsmentioning

confidence: 99%

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Sulfonamide Drugs Binding to the Colchicine Site of Tubulin: Thermodynamic Analysis of the Drug−Tubulin Interactions by Isothermal Titration Calorimetry

et al. 2004

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The discovery of several sulfonamide drugs paved the way toward the synthesis of 6 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide, E7010) and 7 (N-(3-fluoro-4-methoxyphenyl)pentafluorobenzenesulfonamide, T138067), both of which inhibit tubulin polymerization and are under clinical development. A series of diarylsulfonamides containing an indole scaffold was also found to have antimitotic properties, but their mode of interactions with tubulin has remained unidentified so far. In this study, we demonstrate that these sulfonamide drugs bind to the colchicine site of tubulin in a reversible manner. They quenched intrinsic tryptophan fluorescence of tubulin presumably due to drug-induced conformational changes in the protein, but were unable to modulate GTPase activity of tubulin in contrast to colchicine that enhances the same enzymatic activity. Further investigation using isothermal titration calorimetry (ITC) revealed that 5 (N-(5-chloro-7-indolyl)-4-methoxybenzenesulfonamide) afforded a large positive value of heat capacity change (DeltaC(p)() = +264 cal mol(-1) K(-1)) on binding to tubulin, suggesting a substantial conformational transition in the protein along with partial enthalpy-entropy compensation. On the other hand, the 2-chloro regioisomer 2 gave a large negative value of DeltaC(p)() (-589 cal mol(-1) K(-1)) along with complete enthalpy-entropy compensation. This thermodynamic profile was thought to be attributable to a prominent contribution of van der Waals interaction and hydrogen bonding between specific groups in the drug-tubulin complex. These results indicate that a mere alteration in the position of a single substituent chlorine on the indole scaffold has a great influence on the drug-tubulin binding thermodynamics.

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Section: Methodsmentioning

confidence: 99%

“…The reactions were stopped by adding 0.2 mM phenylmethanesulfonyl fluoride (PMSF). Then all samples were analyzed using SDS−PAGE (12% polyacrylamide gel) followed by Coomassie blue staining …”

Section: Methodsmentioning

confidence: 99%

Sulfonamide Drugs Binding to the Colchicine Site of Tubulin: Thermodynamic Analysis of the Drug−Tubulin Interactions by Isothermal Titration Calorimetry

et al. 2004

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“…40 Recently, however, studies from our laboratory have shown that isocolchicine becomes biologically active, binds tubulin with higher affinity, inhibits tubulin self assembly at low drug concentrations, and competes with [ 3 H]-colchicine for binding to tubulin upon introducing suitable hydrophobic groups like NBD or a dansyl moiety at the C-7 position of isocolchicine. 69,70 It has been found that the congeners of isocolchicine family [with hydrophobic substitution on B ring namely NBD-isocolcemid (4j) or NH-dansyl isocolchicine (4k)] have rate constants varying linearly with drug concentrations. The parent molecule, isocolchicine (4e) binds to tubulin in a single step because of its inability to fit itself into an altered second more planar conformation.…”

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%

“…Since the high activation energy of colchicine-tubulin interaction depends upon this second conformational change step, so it can be predicted that NBD-isocolcemid binding to tubulin is also confined to a one-step process. 69 The second step leading to conformational change of both drug and protein is missing. This observation is supported from the absence of GTPase activity and from the identical cleavage pattern of native protein for NBD-isocolcemid (4j)-tubulin complex.…”

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%

“…This observation is supported from the absence of GTPase activity and from the identical cleavage pattern of native protein for NBD-isocolcemid (4j)-tubulin complex. 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.…”

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%

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

Bhattacharyya

Panda

Gupta

et al. 2007

Medicinal Research Reviews

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434

311

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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.

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-NH-Dansyl Isocolchicine Exhibits a Significantly Improved Tubulin-Binding Affinity and Microtubule Inhibition in Comparison to Isocolchicine by Binding Tubulin through Its A and B Rings

et al. 2005

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Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.

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

Cited by 5 publications

References 27 publications

Sulfonamide Drugs Binding to the Colchicine Site of Tubulin: Thermodynamic Analysis of the Drug−Tubulin Interactions by Isothermal Titration Calorimetry

Sulfonamide Drugs Binding to the Colchicine Site of Tubulin: Thermodynamic Analysis of the Drug−Tubulin Interactions by Isothermal Titration Calorimetry

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

-NH-Dansyl Isocolchicine Exhibits a Significantly Improved Tubulin-Binding Affinity and Microtubule Inhibition in Comparison to Isocolchicine by Binding Tubulin through Its A and B Rings

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