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The conformations of two paclitaxel analogs modified at the C-2‘ position, 2‘-deoxypaclitaxel and 2‘-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2‘-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2‘-acetate, an analog in which the 2‘-hydroxyl group hydrogen-bonding capacity has also been eliminated.

The hydrolysis products of taxol have been isolated by high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectroscopy. In contrast to taxol, the major hydrolysis product, baccatin III, has little cytotoxic activity and does not promote in vitro microtubule assembly. In cell culture medium, the concentration of taxol decreases with time and 7-epitaxol, which exhibits properties comparable to those of taxol both on cells and on in vitro microtubuli polymerization, is formed. Baccatin III is found in small quantities in the cell medium, although it is barely detectable within the cells. It is concluded that 7-epitaxol is the major derivative of taxol found in cells and that its presence does not alter, in a major way, the overall biological activity of taxol.

Taxol stabilizes microtubules against the depolymerizing effects of cold temperature, drugs and Ca++. In this report, the effect of alkaline pH on microtubules polymerized in the presence of taxol has been studied. Although taxol-microtubules are more stable than microtubules assembled in the presence of GTP, taxol-microtubules can be partially disassembled when the pH becomes more alkaline. A portion of the recovered tubulin dimer is assembly competent upon pH adjustment to approximately 6.6 and the microtubules formed upon the induction of assembly by GTP are normal as judged by electron microscopy. The data indicate that alkaline pH can be used to recover assembly-competent tubulin from a taxol-microtubule complex. At pH 6.6, taxol-induced polymers consisted of two components. The majority were microtubules, but in addition hoops and ribbons were also present. At alkaline pH, the microtubules were more stable than the hoops and ribbons and at pH greater than 7.5 they were the only stable structures. Microtubules stabilized by taxol are protected against the depolymerizing action of podophyllotoxin even at alkaline pH, whereas the hoops and ribbons are depolymerized.

Staurosporine, a protein kinase inhibitor, induces neurite outgrowth in pheochromocytoma cells and, therefore, may serve as a potential prototype for neurotropic drugs. The principal aim of the present study was to characterize the cytoskeletal properties of neurites induced in pheochromocytoma cells by staurosporine, in comparison to those induced by nerve growth factor, with emphasis on tubulin and tau proteins. Two major findings are described: a) staurosporine rapidly induces outgrowth of neurites that are resistant to colchicine treatment; and b) staurosporine treatment causes a rapid increase in tau protein levels, with a time course similar to the initiation of its neurotropic effects. The following observations exclude tubulin as the cellular target for staurosporine action: a) the level, cellular distribution, and assembly properties of tubulin are not affected by staurosporine treatment; and b) colchicine uptake, its binding to tubulin, and its interference with tubulin polymerization are not changed by staurosporine. On the other hand, staurosporine treatment causes a transient, dose-dependent increase in tau protein levels. This increase, which is already evident after 1 hr, reaches a maximum of 2 to 3 fold after 5 hr of treatment and declines to basal level within the next 10 to 15 hr. The rapid, transient increase of tau protein levels induced by staurosporine is reminiscent of its neurotropic properties. Here we characterize and compare the cytoskeletal properties of neurites induced by treatment with staurosporine and with nerve growth factor, and we offer a mechanistic explanation for the rapid stabilization of staurosporine induced neurites.