guanosine-triphosphate and discodermolide

To determine whether inhibitors of microtubule assembly inhibit polymerization induced by discodermolide and epothilone B, as well as paclitaxel, and to quantitatively measure such effects.. Inhibition was quantitated by measuring polymer formation either by turbidimetry or by centrifugation, and the amount of inhibitor required to inhibit 50% relative to an appropriate control reaction was determined.. The inhibitory drugs evaluated were four colchicine site agents (combretastatin A-4, podophyllotoxin, nocodazole, and N-acetylcolchinol- O-methyl ether), maytansine, which competitively inhibits the binding of Catharanthus alkaloids to tubulin, halichondrin B and phomopsin A, which noncompetitively inhibit the binding of Catharanthus alkaloids to tubulin, and the depsipeptide dolastatin 15. While relative inhibitory effects were highly variable, a few broad generalizations can be made. First, assembly reactions that were either enhanced or dependent upon all three stimulatory drugs were subject to inhibition by all inhibitors. Second, the more readily the tubulin assembled, the greater the concentration of inhibitor required to inhibit polymerization. Drug IC50 values were generally lowest with no stimulatory drug and highest when discodermolide was present; IC50 values were higher as reaction temperature increased; and IC50 values were higher as the tubulin concentration increased. Third, inhibition of assembly by inhibitors of Catharanthus alkaloid binding to tubulin changed much less as a function of changes in reaction conditions than inhibition by inhibitors of colchicine binding.. Since there was no apparent quantitative predictability of combined drug interactions with tubulin, any combination of interest must be studied in detail.

Topics: Alkanes; Carbamates; Dose-Response Relationship, Drug; Epothilones; Guanosine Triphosphate; Lactones; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Polymers; Pyrones; Tubulin; Tubulin Modulators

Computer-assisted structure analysis indicated (+)-discodermolide, a polyhydroxylated alkatetraene lactone marine natural product, was an antimitotic compound, and we confirmed this prediction. Previous work had shown an accumulation of discodermolide-treated cells in the G2/M portion of the cell cycle, and we have now found that discodermolide arrests Burkitt lymphoma cells in mitosis. Discodermolide-treated breast carcinoma cells displayed spectacular rearrangement of the microtubule cytoskeleton, including extensive microtubule bundling. Microtubule rearrangement that occurred with 10 nM discodermolide required 1 microM taxol. Discodermolide had equally impressive effects on tubulin assembly in vitro. Near-total polymerization occurred at 0 degree C with tubulin plus microtubule-associated proteins (MAPs) under conditions in which taxol at an identical concentration was inactive. Without MAPs and/or without GTP, tubulin assembly was also more vigorous with discodermolide than with taxol under every reaction condition examined. Discodermolide-induced polymer differed from taxol-induced polymer in that it was completely stable at 0 degree C in the presence of high concentrations of Ca2+. In a quantitative assay designed to select for agents more effective than taxol in inducing assembly, discodermolide had an EC50 value of 3.2 microM versus 23 microM for taxol.

Topics: Alkanes; Animals; Antineoplastic Agents; Breast Neoplasms; Burkitt Lymphoma; Calcium; Carbamates; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Fluorescent Antibody Technique, Indirect; Guanosine Triphosphate; Humans; Kinetics; Lactones; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Porifera; Pyrones; Time Factors; Tubulin; Tumor Cells, Cultured