Baccatin III Induces Assembly of Purified Tubulin into Long Microtubules

Abstract

Baccatin III is widely considered to be an inactive derivative of Taxol. We have reexamined its effect on in vitro assembly of tubulin under a variety of conditions. We found baccatin III to be active in all circumstances in which Taxol is active: it assembled GTP-tubulin, GDP-tubulin, and microtubule protein into normal microtubules and stabilized these polymers against cold-induced disassembly. The effect of baccatin III on in vitro microtubule assembly was quantitatively assessed through determination of critical concentrations, which can be used to obtain the apparent equilibrium constants for the addition of tubulin subunits to growing microtubules. The apparent equilibrium constants for the growth reaction for baccatin III-induced GTP-tubulin and GDP-tubulin assembly measured at 37 degrees C were 4.2-4.6-fold less than those measured for Taxol-induced GTP-tubulin and GDP-tubulin assembly. These data indicate that the entire Taxol side chain contributes only about -1 kcal/mol to the apparent standard free energy of microtubule growth at 37 degrees C regardless of the nature of the E site nucleotide. These data also support the idea that the majority of the interactions between Taxol and tubulin that affect this equilibrium occur between the baccatin portion of the molecule and the binding site. We have also observed a structural difference in microtubules formed using baccatin III and Taxol. Baccatin III-induced microtubules were routinely much longer than those assembled by Taxol, even when very high concentrations of baccatin III were employed. One interpretation of these data is that baccatin III and Taxol differ in their abilities to nucleate GTP-tubulin. This difference in activity may have bearing on the large disparity in cytotoxicity of the two molecules.

Relationship between the Structures of Taxane Derivatives and Their Microtubule Polymerization Activity

“…These findings strongly support the hypothesis of Chatterjee and coworkers that the C-13 side chain is not an absolute requirement for the taxane molecule. 14) Furthermore, our findings on taxanes 3, 6, and 7 also suggest that the C-2 benzoyl group is not indispensable for a promotional effect on tubulin assembly.…”

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

“…However, increased apoptotic (hypoploid) sub G1 cells after fungal baccatin III treatment occurred at higher concentration compared to fungal taxol. This might be either due to the high affinity of taxol to microtubules [40] or involvement of non-tubulin factors [41]. Under the conditions used in this study, both fungal taxol and baccatin III induce apoptotic cell death in JR4-Jurkat, HepG2, HeLa, Ovcar3 and T47D cells with very similar kinetics, as determined by the appearance of hypoploid DNA, although the concentrations of fungal taxol varied from 10 nM for T47D, JR4-Jurkat and HeLa cells, to 100 nM for HepG2 and Ovcar3 cells.…”

Synthesis and bioactivity of a side chain bridged paclitaxel: A test of the T-Taxol conformation

“… a Disassociation constants (Kd) determined as described in Supporting Information. b Cytotoxicity determined as described in Supporting Information. c Data from ref 36. …”

“…Constrained cyclic carbamate 4 , open chain carbamate 5a , and PTX were evaluated for tubulin-affinity, tubulin – assembly activity,36 and antiproliferative activity against the A2780 and PC3 cell lines (Table 1). Association constants for PTX and 4 binding to GTP-microtubules were assessed by a fluorescent competition assay as described in the Supporting Information.…”