Probing the Environment of Tubulin-Bound Paclitaxel Using Fluorescent Paclitaxel Analogues
- 1 April 1997
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (17), 5179-5184
- https://doi.org/10.1021/bi962891m
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 π 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.Keywords
This publication has 7 references indexed in Scilit:
- Kinetics of Association of Myosin Subfragment-1 to Unlabeled and Pyrenyl-labeled ActinJournal of Biological Chemistry, 1996
- Synthesis, Conformational Analysis, and Biological Evaluation of Heteroaromatic TaxanesThe Journal of Organic Chemistry, 1996
- Selective Deesterification Studies on Taxanes: Simple and Efficient Hydrazinolysis of C-10 and C-13 Ester FunctionalitiesThe Journal of Organic Chemistry, 1995
- NMR and molecular modeling study of active and inactive taxol analogues in aqueous and nonaqueous solutionCanadian Journal of Chemistry, 1994
- "Hydrophobic collapse" of taxol and Taxotere solution conformations in mixtures of water and organic solventJournal of the American Chemical Society, 1993
- Novel Biologically Active Taxol Analogues: Baccatin III 13-(N-(p-chlorobenzoyl)-(2′R,3′S)-3′-phenylisoserinate) and Baccatin III 13-(N-benzoyl-(2′R,3′S)-3′-(p-chlorophenyl)isoserinate)Bioorganic & Medicinal Chemistry Letters, 1992
- The effects of dimethyl sulfoxide on the kinetics of tubulin assemblyBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988