TAXOL--MICROTUBULE PHYSICAL INTERACTIONS

紫杉烷类化合物是一类重要的新化合物， 癌症治疗的承诺。 紫杉醇（紫杉醇），原型，是一种 具有显著活性的复合二萜类天然产物， 难治性卵巢癌和乳腺癌。 泰索帝（多西他赛），a 半合成类似物，正在进行高级临床试验， 许多新的活性紫杉烷处于不同的开发阶段。 的 紫杉烷作用的亚细胞靶点是微管（MT），但 作用机制是独特的，涉及稳定，而不是 微管的分解紫杉烷相互作用的马内细节 微管未知。紫杉醇与组装的MT结合，但不与 微管蛋白异二聚体结合研究显示，在 大约1：1（摩尔：摩尔）紫杉醇：微管蛋白异二聚体。 光亲和 研究发现β微管蛋白上的两个肽域可能是紫杉醇 接触位点：N-末端33个氨基酸和残基217-231。 大多数研究表明光标记的β微管蛋白单独;一些显示α tubulin标记。 β微管蛋白的分子结构和 α/β微管蛋白相互作用的细节是未知的。 因此 单个紫杉烷分子接触两种肽的可行性 同时，不清楚。 也不知道是否一个单一的紫杉烷 分子可以同时与α和β-微管蛋白相互作用。 最近的溶液构象研究表明，紫杉醇在疏水和 水性环境采用“堆叠”构象。 疏水 力和网状分子间氢键（涉及两个 紫杉烷环系统和C13侧链）稳定结构。 的 堆栈构象是服从无限传播的加法 紫杉烷单体的堆叠端，因此可以桥接 相当远的距离。 泰索帝具有类似的形成堆叠的能力， 但无活性的紫杉烷（巴卡亭III）不具有这种活性。 我们将调查 紫杉醇在MT存在下的构象，并验证假设 紫杉烷与微管的结合涉及紫杉烷：紫杉烷相互作用。 将合成荧光紫杉烷来探测微管相互作用 低浓度。 CD（圆二色性）、荧光和NMR 光谱学是探测紫杉烷的主要工具 在MT存在下的构象，最终目的是提供 紫杉烷-微管相互作用的分子细节。初步CD 结果表明，独特的光谱特征的疏水本地化 紫杉醇在MT的存在下。紫杉烷：MT相互作用的解析 细节对于理解MT结构有很大的相关性 和功能，并为合理设计新的紫杉烷。

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.