RP 56976 (taxotere), a new semisynthetic analogue of taxol, is a potentially important chemotherapeutic agent for the treatment of cancer. We report here that this drug is a potent inhibitor of cell replication and, like taxol, promotes the in vitro assembly of stable microtubules in the absence of guanosine triphosphate and induces microtubule-bundle formation in cells. Compared with taxol, RP 56976 is slightly more active as a promoter of tubulin polymerization. As an inhibitor of cell replication, RP 56976 is 2.5-fold more potent than taxol in J774.2 and P388 cells and at least 5-fold more potent in taxol-resistant cells.

In many biochemical processes large biomolecular assemblies play important roles. X-ray scattering is a label-free bulk method that can probe the structure of large self-assembled complexes in solution. As we demonstrate in this paper, solution X-ray scattering can measure complex supramolecular assemblies at high sensitivity and resolution. At high resolution, however, data analysis of larger complexes is computationally demanding. We present an efficient method to compute the scattering curves from complex structures over a wide range of scattering angles. In our computational method, structures are defined as hierarchical trees in which repeating subunits are docked into their assembly symmetries, describing the manner subunits repeat in the structure (in other words, the locations and orientations of the repeating subunits). The amplitude of the assembly is calculated by computing the amplitudes of the basic subunits on 3D reciprocal-space grids, moving up in the hierarchy, calculating the grids of larger structures, and repeating this process for all the leaves and nodes of the tree. For very large structures, we developed a hybrid method that sums grids of smaller subunits in order to avoid numerical artifacts. We developed protocols for obtaining high-resolution solution X-ray scattering data from taxol-free microtubules at a wide range of scattering angles. We then validated our method by adequately modeling these high-resolution data. The higher speed and accuracy of our method, over existing methods, is demonstrated for smaller structures: short microtubule and tobacco mosaic virus. Our algorithm may be integrated into various structure prediction computational tools, simulations, and theoretical models, and provide means for testing their predicted structural model, by calculating the expected X-ray scattering curve and comparing with experimental data.

Single and double tubulin rings were studied under a range of conditions and during microtubule (MT) assembly and disassembly. Here, tubulin was purified from porcine brain and used without any further modifications or additives that promote ring assembly. The structure of single GDP-rich tubulin rings was determined by cryo-transmission electron microscopy and synchrotron solution X-ray scattering. The scattering curves were fitted to atomic models, using our state-of-the-art analysis software, . We found that there is a critical concentration for ring formation, which increased with GTP concentration with temperature. MT assembly or disassembly, induced by changes in temperature, was analyzed by time-resolved small-angle X-ray scattering. During MT assembly, the fraction of rings and unassembled dimers simultaneously decreased. During MT disassembly, the mass fraction of dimers increased. The increase in the concentration of rings was delayed until the fraction of dimers was sufficiently high. We verified that pure dimers, eluted via size-exclusion chromatography, could also form rings. Interestingly, X-ray radiation triggered tubulin ring disassembly. The concentration of disassembled rings versus exposure time followed a first-order kinetics. The disassembly rate constant and initial concentration were determined. X-ray radiation-triggered disassembly was used to determine the concentration of rings. We confirmed that following a temperature jump, the mass fraction of rings decreased and then stabilized at a constant value during the first stage of the MT assembly kinetics. This study sheds light on the most basic assembly and disassembly conditions for in vitro single GDP-rich tubulin rings and their relation to MT kinetics.

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.

This observation is the first step in a study to determine the binding site for taxol on the microtubule.