Probing the Environment of Tubulin-Bound Paclitaxel Using Fluorescent Paclitaxel Analogues

Abstract

To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- and N- as well as the 3'-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3'-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of the N-meta derivative to tubulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11889-11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to the N-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- and N-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to pi stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- and N-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- and N-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.

Fluorescent taxoids as probes of the microtubule cytoskeleton

“…Interestingly, the change detected in the fluorescein absorption spectrum upon FLUTAX binding to microtubules is evidence of a cationic microenvironment of the fluorescein group attached to position 7 of taxol, compatible with the proximity of a metal cation or of some basic residues of tubulin, even the protein as a whole has a net negative charge. In this context, fluorescent N(dimethylamino)benzoyl taxol derivatives at positions 7 and 10 have been reported to probe a less hydrophobic environment in microtubules than 2-and N-(derivatives) [Sengupta et al, 1997].…”

“…For a comparison with the weakly active nitrobenzoxadiazol derivatives of docetaxel at positions 7 and 3Ј, see Dubois et al [1995]. A very weak visualization of some spindles was obtained with CU-TAX [Souto et al, 1995] employing near-UV fluorescence microscope optics, which was not observed with the biochemical probe 2-debenzoyl-2-(m-aminobenzoyl)taxol [Han et al, 1996]; microtubule visualization with (dimethylamino)benzoyl taxoid probes [Sengupta et al, 1997] has not been documented. Therefore, whereas we have obtained several active green fluorescent taxoids and have employed them for the first time to label cellular microtubules specifically, efficiently and accurately, comparable red and blue fluorescent taxoids are not yet available.…”

Docking and hydropathic scoring of polysubstituted pyrrole compounds with antitubulin activity

“…The solvent accessible surface area for the amide nitrogens of the protein backbone were corrected with the "+20" option. Finally, HINT scores were plotted against experimental binding free energies that were calculated using the standard Gibbs free energy equation: (4) where R is Boltzmann's constant (1.9872 cal K −1 mol −1 ) and T is 298 K; K eq is an equilibrium binding constant, ideally K D . In this work measured IC 50 values are being used as approximations for equilibrium constants.…”

“…Colchicine, the first drug known to bind to the tubulin protein [1,2], inhibits microtubule formation and causes loss of cellular microtubules. In contrast, paclitaxel and its analogues actually promote microtubule polymer formation [3][4][5], albeit by acting at a different site on tubulin than colchicine. A variety of small molecules with diverse molecular scaffolds have been shown to bind tubulin at the colchicine site [6][7][8][9].…”

The bioactive Taxol conformation on β-tubulin: Experimental evidence from highly active constrained analogs

“…Thus, photoaffinity labeling has shown that a 3Ј-(p-azidobenzamido)Taxol derivative labels the Nterminal 31 amino acids of ␤-tubulin (15), whereas 2-(mazidobenzoyl)Taxol labels residues 217-231 of ␤-tubulin (16), and a C-7 benzophenone derivative labeled Arg 282 in ␤-tubulin (17). Fluorescence spectroscopy has yielded valuable information (18)(19)(20), but the most important results to date have come from the recently determined 3.7-Å structure of the ␣␤-tubulinTaxol complex obtained by electron crystallography of zincinduced tubulin sheets (21,22). Although this structure shows the location of the binding site on ␤-tubulin, it does not enable the conformation of the ligand to be determined with precision.…”