Interaction of a fluorescent paclitaxel analog with tubulin

Abstract

To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3-(dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel-binding site on tubulin.

Characterization of nuclear βII‐tubulin in tumor cells: A possible novel target for taxol

“…In addition, the nucleoli were stained by flutax-2, suggesting that tubulin binding to flutax-2 was enriched in the nucleoli though taxol binds to soluble tubulin dimers with a reduced affinity [Parness and Horwitz, 1981;Takoudju et al, 1988;Diaz et al, 1993;Sengupta et al, 1995]. This result is consistent with the fluorescein-colchicine staining of the nuclear matrix in C6 cells, in which an accumulation of tubulin dimers in the nucleolus was observed (Fig.…”

The Tubulin Binding Mode of MT Stabilizing and Destabilizing Agents Studied by NMR

“…DMAT competes with PTX for binding to MT and is almost as effective as PTX in the MT-assembly assay. When binding to dimeric tubulin under non-assembly conditions, DMAT displays much lower affinity than for MT ( K d 49 ± 8 m M at 25°C) [115] , that is of the same order as that proposed for EpoA in the NMR study. Weak binding of EpoA to soluble tubulin has also been postulated as the first step of one of the possible mechanisms of EpoA-induced MT assembly [114] .…”

“…This dissociation constant of EpoA for nonpolymerized tubulin is significantly larger than that for MT ( K d 34 ± 4 nM at 37°C [114] ). Binding of MT-stabilizing agents to nonpolymerized tubulin had been suggested in a previous study with the fluorescent PTX analogue DMAT [115] . DMAT competes with PTX for binding to MT and is almost as effective as PTX in the MT-assembly assay.…”

Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification

“…To address such issues, structural manipulations that yield valuable properties such as fluorescence are often explored. One prominent example of this approach is the design and creation of fluorescent analogs of paclitaxel (Taxol), which have proven useful for defining the mechanistic details of tubulin interactions (Sengupta et al, 1995;Díaz et al, 2000). The development of fluorescent compounds is worthwhile only if such compounds retain the activity of the class of compounds being investigated.…”