(-DISCODERMOLIDE BINDS TO MICROTUBULES IN STOICHIOMETRIC RATIO TO TUBULIN DIMERS, BLOCKS TAXOL BINDING AND RESULTS IN MITOTIC ARREST())

Background: The marine natural product (+)-discodermolide has potent i mmunosuppressive activity. It inhibits proliferation of a wide range o f human and murine cells, induces cell cycle arrest in the G2 or M pha se and was recently shown to stabilize microtubules. Total synthesis o f discodermolide has made it possible to generate variants of the comp ound to study its intracellular function in detail. Results: We have d etermined that (+)-discodermolide arrests MG63 cells at M phase, and h as a stabilizing effect on microtubules. in vitro studies show that di scodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dime rs in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-po lymerized microtubules at near stoichiometric level, whereas taxol bin ds discodermolide-induced microtubules poorly. Competition data show t hat the binding of microtubules by discodermolide and taxol are mutual ly exclusive; discodermolide binds with higher affinity than taxol. Th e results of binding assays carried out in vivo or in cell lysates als o suggest that the microtubule network is discodermolide's cellular ta rget. Conclusions: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabil izing effect on microtubules. In vitro, discodermolide polymerizes pur ified tubulin potently in the absence of MAPs. It binds microtubules a t one molecule per tubulin dimer with a higher affinity than taxol, an d the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding acti vity that is consistent with its effects on microtubules.