TAXOL--MICROTUBULE PHYSICAL INTERACTIONS

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The taxanes are an important new class of compounds showing great promise for cancer therapy. Taxol (paclitaxel), the prototype, is a complex diterpenoid natural product that has significant activity in refractory ovarian and breast cancer. Taxotere (docetaxel), a semisynthetic analogue, is in advanced clinical trial, and a large number of new active taxanes are in various stages of development. The subcellular target of taxane action is microtubules (MT), but the mechanism of action is unique, involving stabilization, rather than disassembly of microtubules. Mane details of taxane interaction with microtubules are unknown. Taxol binds to assembled MT, but not to tubulin heterodimer. Binding studies show maximal effects on MT at approximately 1:1 (mole:mole) taxol:tubulin heterodimer. Photoaffinity studies identified two peptide domains on beta tubulin as possible taxol contact sites: the N-terminal 33 amino acids, and residues 217-231. Most studies show photolabeling of beta tubulin alone; some show alpha tubulin labeling also. The molecular structure of beta tubulin and details of alpha/beta tubulin interaction are unknown. Thus the feasibility of a single taxane molecule contacting both peptides simultaneously is unclear. Nor is it known whether a single taxane molecule could interact simultaneously with both alpha and beta-tubulin. Recent solution conformation studies show that taxol in hydrophobic and aqueous environments adopts a "stacked" conformation. Hydrophobic forces and a mesh of net-like intermolecular H-bonds (involving both the taxane ring system and the C13 side chain) stabilize the structure. The stack conformation is amenable to infinite propagation by the addition of taxane monomer at the stack ends, and therefore could bridge considerable distances. Taxotere has a similar ability to form stacks, but inactive taxanes (Baccatin III) do not. We will investigate the conformation of taxol in the presence of MT, and test the hypothesis that taxane binding to microtubules involves taxane:taxane interaction. Fluorescent taxanes will be synthesized to probe microtubule interaction at low concentration. CD (Circular Dichroism), fluorescence, and NMR spectroscopy are the primary tools to be used to probe taxane conformation in the presence of MT, with the ultimate aim of providing molecular details of taxane-microtubule interaction. Preliminary CD results show the unique spectral signature of hydrophobically-localized taxol in the presence of MT. Elucidation of taxane: MT interaction details would have considerable relevance to understanding MT structure and function, and for the rational design of new taxanes.