sarcodictyin-a and eleutherobin

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.

Topics: Actin Cytoskeleton; Alkaloids; Amino Acid Sequence; Animals; Dimerization; Diterpenes; Docetaxel; Epothilones; Epoxy Compounds; Excipients; Guanosine Triphosphate; Microtubules; Molecular Sequence Data; Paclitaxel; Protein Conformation; Surface Properties; Swine; Taxoids; Thiazoles; Tubulin

We examined interactions with purified tubulin of synthetic sarcodictyins A and B and eleutherobin (coral-derived antimitotic agents) and of compound 1, an analogue of sarcodictyin A methylated at the C-3 oxygen atom (i.e., the methyl ketal analogue of sarcodictyin A and thus structurally similar to eleutherobin but lacking the C-3 sugar moiety). Eleutherobin was much more active than sarcodictyins A and B, which were somewhat more active than compound 1. Effects of eleutherobin did not differ greatly from those of paclitaxel and epothilone A. Eleutherobin and epothilone A were competitive inhibitors of the binding of radiolabeled paclitaxel to tubulin polymer (apparent Ki values of 2.1 and 2.6 microM, respectively). Tubulin assembly reactions induced by all compounds were similar to the paclitaxel-driven reactions in being enhanced by the addition of microtubule-associated proteins and/or GTP to the reaction mixture and by progressively higher reaction temperatures. Antiproliferative activity was studied in six human cancer cell lines, including two paclitaxel-resistant lines with point mutations in a beta-tubulin gene. Except for compound 1, effects on cell growth were generally in accord with effects on purified tubulin. Thus, sarcodictyins A and B had IC50 values in the 200-500 nM range; paclitaxel, <10 nM (except in the resistant lines); and eleutherobin and epothilone A, 10-40 nM. The antiproliferative activity of compound 1 was more comparable to that of eleutherobin than sarcodictyin A, despite its weak interaction with tubulin. The activities of the sarcodictyins, eleutherobin, and compound 1 in the mutant ovarian lines were similar to their activities in the parental line.

Topics: Alkaloids; Animals; Antineoplastic Agents; Binding, Competitive; Cnidaria; Diterpenes; Docetaxel; Epothilones; Epoxy Compounds; Growth Inhibitors; Guanosine Triphosphate; Humans; Microtubule-Associated Proteins; Paclitaxel; Polymers; Protein Binding; Protein Processing, Post-Translational; Taxoids; Thiazoles; Tubulin; Tumor Cells, Cultured

Topics: Alkaloids; Antineoplastic Agents; Diterpenes; Molecular Mimicry; Paclitaxel; Structure-Activity Relationship