PROBING THE ENVIRONMENT OF TUBULIN-BOUND PACLITAXEL USING FLUORESCENTPACLITAXEL ANALOGS

TO determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent an alogues in which a (dimethylamino)benzoyl group has been introduced in to 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 func tions. In a tubulin assembly assay, the N-m- and N-p-(dimethylamino)be nzoyl derivatives had activities comparable to the activity of paclita xel. 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 hydro gen bonds with protic solvents. But the 7- and 10-substituted analogue s appear to be more affected by protic solvents than the other analogu es. Previously, we studied the binding of the N-meta derivative to tub ulin and microtubules [Sengupta, S., et al. (1995) Biochemistry 34, 11 889-11894]. 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 de rivatives bound. The 2- and N-substituted analogues bind to microtubul es with an increase in fluorescence intensity over that which was obse rved with tubulin, whereas binding of the 7- and 10-substituted analog ues was accompanied by a large quenching in fluorescence. This quenchi ng 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 g roups with an aromatic side chain. The presence of paclitaxel with mic rotubules prevented the fluorescence increase of the 2- and N-derivati ves and quenching of the 7- and 10-derivatives. The difference in beha vior 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.