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The biology of three novel natural product microtubule interacting agents : ceratamine A and B and dimethyl varacin

University of British Columbia

Microtubules are dynamic polymers of the protein tubulin. The dynamic property of microtubules allows the network to breakdown and form new structures like the mitotic spindle. Microtubule associated proteins and small molecules can alter the dynamics. Changing the dynamics by the addition of small molecules can prevent the proper formation of the mitotic spindle and lead to mitotic arrest. Some of these antimitotic agents have had clinical success in cancer treatment with the vinca alkaloids inducing the underpolymerisation and paclitaxel (Taxol™) promoting the overpolymerisation of microtubules. In addition, small molecules that alter the microtubule network are gaining use in chemical genetics. A phenotypic antimitotic assay has been used to discover three novel compounds that interact with the microtubule network. The first two, ceratamine A and B are classic antimitotic agents that block cells in M-phase and prevent proliferation, probably by disrupting the microtubule network. They stimulate the over-polymerization of microtubules in vitro as determined by both microtubule polymerization assays and electron microscopy. In vivo, ceratamines induce the formation of tubulin-containing structures not previously described. These structures include pillars of tubulin in mitotic cells and a perinuclear microtubule network during interphase. Ceratamines do not compete with paclitaxel for binding to the microtubules, and are very structurally simple. The same phenotypic antimitotic assay was used to discover dimethyl varacin (DMV). Flow cytometry data indicated that DMV is not an antimitotic agent and does not block cells in any particular phase of the cell cycle, though it does inhibit proliferation. It induces strong GF-7 phospho-specific antibody binding (typical only in mitosis) from all phases of the cell cycle. Western blots and Kinexiis Kinetworks™ screens showed that a two hours DMV treatment at 5 μM leads to the strong activation of MAPK pathways (Erkl/2, p38 α MAPK, and JNK/SAPK) without a significant increase in Cdc2 activity or global phosphorylation. It was also found to strongly inhibit microtubule formation in vitro and in vivo. Though both DMV and the ceratamines were discovered in the same antimitotic screen and both target microtubules, they have dramatically different biological properties.

Thesis/Dissertation

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2009-12-03

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http://hdl.handle.net/2429/16296

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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