TAXOL--MICROTUBULE PHYSICAL INTERACTIONS

紫杉烷是一类重要的新化合物，具有很好的应用前景。 对癌症治疗的承诺。紫杉醇(紫杉醇)的原型是一种 具有显著活性的复杂二萜类天然产物 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，并有很大的 一些新的活性紫杉烷正处于不同的开发阶段。这个 紫杉烷作用的亚细胞靶点是微管，但 作用机制是独一无二的，涉及稳定，而不是 微管的解体。与紫杉烷相互作用的MANE细节 微管是未知的。紫杉醇与组装的MT结合，但不结合 微管蛋白杂二聚体。结合研究显示对MT的影响最大 紫杉醇：微管蛋白杂二聚体的摩尔比约为1：1。光亲和力 研究确定β-微管蛋白上的两个肽段可能是紫杉醇 接触部位：N端33个氨基酸，残基217-231。 大多数研究只显示了β微管蛋白的光标记；一些研究显示了阿尔法 微管蛋白标记也是如此。β微管蛋白和β-微管蛋白的分子结构 α/β微管蛋白相互作用的细节尚不清楚。因此， 一种紫杉烷分子与两种多肽结合的可行性 同时也不清楚。也不知道一种单一的紫杉烷 分子可同时与α微管蛋白和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 水环境采用“堆积”构象。疏水性 作用力和网状分子间氢键(涉及 紫杉烷环系和C13侧链)稳定结构。这个 堆栈的构象可以通过加法无限传播 在堆叠末端的紫杉烷单体，因此可以桥接 相当远的距离。Taxotere具有类似的形成堆叠的能力， 但非活性紫杉烷(巴卡菌素III)则不起作用。我们将调查 紫杉醇在MT存在下的构象，并验证该假说 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 在低浓度下。圆二色谱、荧光和核磁共振 光谱是探测紫杉烷的主要工具 在MT存在的情况下进行整合，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，疏水局域结构具有独特的光谱特征 紫杉醇在MT存在下。紫杉烷的阐明：MT相互作用 细节将与理解MT结构有相当大的相关性 和功能，以及新紫杉烷的合理设计。

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.