ABSTRACT
Taxol is a structurally novel and clinically effective antitumor drug, which, unlike other antimitotic agents, induces the assembly of tubulin into microtubules. To characterize the binding site(s) of taxol on the microtubule, taxol-based photoaffinity reagents 1 and 2 bearing photoreactive groups on the A-ring side chain were prepared and evaluated. Taxol analogue 1 exhibits better microtubule assembly activity, greater cytotoxicity toward J774.2 cells, and more specific and efficient photolabeling of the beta-subunit of tubulin than does analogue 2. Therefore, it would appear that 1 is the better candidate for further studies aimed at the characterization of the taxol binding site on the microtubule.
Subject(s)
Affinity Labels/pharmacology , Azides/pharmacology , Paclitaxel/analogs & derivatives , Taxoids , Affinity Labels/chemical synthesis , Affinity Labels/metabolism , Animals , Azides/chemical synthesis , Azides/metabolism , CHO Cells , Cattle , Cell Division/drug effects , Cell Line , Cricetinae , Cricetulus , Mice , Microtubules/drug effects , Microtubules/metabolism , Paclitaxel/chemical synthesis , Paclitaxel/metabolism , Paclitaxel/pharmacology , PhotochemistryABSTRACT
Taxol possesses an unusual chemical structure, a unique mechanism of action, and demonstrated activity in human malignancies. It is the only antitumor agent that has a binding site on the microtubule polymer. The interaction of Taxol with the microtubule polymer results in the formation of stable bundles of cellular microtubules that are resistant to depolymerization. Although it has become evident that the microtubule, specifically beta-tubulin, is the target for Taxol, no information is available on the binding site for the drug. In this report, we demonstrate that 3'-(p-azidobenzamido)taxol, an analogue with similar biological activities as Taxol, covalently binds to the N-terminal domain of beta-tubulin after irradiation of the microtubule-drug complex. Taxol competes with [3H]3'-(p-azidobenzamido)-taxol binding, suggesting that the photoaffinity analog and Taxol are binding at the same or overlapping sites. Formic acid cleavage of [3H]3'-(p-azidobenzamido)-taxol-photolabeled beta-tubulin and subsequent protein sequence and mass analyses have identified the N-terminal 31 amino acids as the major site for [3H]3'-(p-azidobenzamido)taxol photoincorporation.
Subject(s)
Affinity Labels/metabolism , Azides/metabolism , Paclitaxel/analogs & derivatives , Taxoids , Tubulin/chemistry , Tubulin/metabolism , Animals , Brain/metabolism , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Microtubule Proteins/isolation & purification , Molecular Weight , Paclitaxel/metabolism , Tritium , Tubulin/isolation & purificationABSTRACT
The antimitotic agent taxol was tested for toxicity towards fungi from different taxonomic groups and found to be particularly active against oomycete fungi. In germinating zoospore cysts of the oomycete Phytophthora capsici the mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on microtubules. Various taxol analogues with deleted A-ring C-13 side chain substituents were tested for toxicity towards P. capsici and Aphanomyces cochlioides to provide insight into structural features required for activity. The importance of the side chain was shown by the much lower activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported previously on mammalian microtubule assembly.
Subject(s)
Antifungal Agents/pharmacology , Fungi/drug effects , Paclitaxel/pharmacology , Cell Division/drug effects , Paclitaxel/analogs & derivatives , Paclitaxel/toxicity , Species Specificity , Structure-Activity RelationshipABSTRACT
Taxol, a potent inhibitor of cell replication, enhances the assembly of tubulin into stable microtubules and promotes the formation of microtubule bundles in cells. In addition to its unique mechanism of action, taxol exhibits unusual promise as an antitumor agent, but its application in cancer chemotherapy is hampered by its limited availability. In order to better define the structure-activity profile of taxol for the design of more accessible drugs and to provide insight into the chemical features of the taxol-microtubule interaction, taxol analogues 3-8, with deleted A-ring side chain substituents and both R and S C-2' configurations, were synthesized from baccatin III through esterification at the hindered 13-hydroxyl. Employing an improved hydroxyl protection strategy, lactate analogues 3 and 4 were prepared with reasonable efficiency owing to their simple side-chain structures, while N-benzoylisoserine analogues 7 and 8 were synthesized through esterification reactions whose rates were enhanced greatly by the participation of the amide functionality. Although less biologically active than taxol, analogues 5-7 were found to promote the polymerization of tubulin and to be cytotoxic; 5 and 6 were considerably more effective than 7, whereas 3, 4, and 8 were least active. Interestingly, tubulin polymerization was sensitive to the C-2' configuration only when the amide substituent was present in the side chain. This observation suggests that the 3'-amide substituent plays an important role in preorganizing the taxol side chain to bind to microtubules.
